ES2727836T3 - RSV immunization regimen - Google Patents

Abstract

An inductive composition of an immune response against respiratory syncytial virus (RSV) that provides one or more RSV antigens for use in a procedure to provide protective immunity against RSV or the protection of a disease caused by RSV in a baby, wherein the procedure comprises administering to the baby at 4 months of age or younger, the inductive composition of the immune response directed against RSV, wherein said baby was a newborn of a woman to whom a composition was administered which provides one or more RSV antigens that induced an immune response directed against RSV during the time said woman was pregnant with said baby, in which the one or more RSV antigens comprise a RSV F glycoprotein.

Description

DESCRIPTION

RSV immunization regimen

Related Requests

This application claims the benefit of US patent application No. 61 / 436,355, filed on January 26, 2011.

SEQUENCE LIST

This application contains a list of sequences that have been submitted in ASCII format via EFS-Web. This ASCII copy, created on Wednesday, January 25, 2012, is called 85448116.txt and is 15,682 bits in size.

Background

The respiratory syncytial virus (RSV) is a non-segmented negative strand RNA envelope of the family Paramyxoviridae, genus Pneumovirus. It is the most common cause of bronchiolitis and pneumonia among children in their first years of life. RSV also produces repeated infections that include serious diseases of the lower respiratory tract, which can occur at any age, especially those with compromised cardiac, pulmonary, or immune systems.

In order to infect a host cell, paramyxoviruses such as RSV, like other enveloped viruses such as the influenza virus and HIV, require the fusion of the viral membrane with the membrane of a host cell. For RSV, the conserved fusion protein (RSV F) fuses with the viral and cellular membranes by coupling to the irreversible refolding of the protein with juxtaposition. In current models based on studies with paramyxovirus, the VRS F protein initially folds into a metastable "prefusion" conformation. During cell entry, the prefusion conformation undergoes refolding and conformational changes in its stable "post-fusion" conformation.

The VRS F protein is translated from the mRNA into a protein of approximately 574 amino acids designated F ⁰ . Post-translational processing of F ⁰ includes the removal of a signaling peptide at the N-terminus by means of a signaling peptidase in the endoplasmic reticulum. Fo is also cleaved at two sites (approximately 109/110 and approximately 136/137) by cellular proteases (in particular furin) in the trans-Golgi. This cleavage results in the elimination of a short intervening sequence and generates two subunits F ¹ (~ 50 kDa; End C; approximately residues 137-574) and F ² (~ 20 kDa; End N; approximately residues 1-109 ) that remain associated with each other. F ¹ contains a hydrophobic fusion peptide at its N-terminus and also two amphipathic septet repeat regions (HRA and HRB). HRA is close to the fusion peptide and HRB is close to the transmembrane domain. three heterodimers F ¹ -F ² is assembled as homotymers of F ¹ -F ² in the virion. A suitable vaccine for babies against RSV infection is currently not available, but is desired.

Previous attempts at vaccines for RSV have led to failed clinical trials in which the vaccine does not protect against infection, and in fact, was associated with an increased risk of severe RSV disease when the vaccinated child became infected. Kim, H.W., et al., Am. J. Epidemiol., 89: 422-434 (1969). Traditional immunization procedures for vaccinating babies are not available. Englund J et al. Vaccine 16 (1998) 1456-1463 describes the protection of babies against certain immunizations by immunization of the mother during pregnancy, and indicates that this is being considered for RSV, after which the immunization of older babies with live attenuated vaccines.

Therefore, improved RSV immunization regimens are necessary.

Summary

The invention relates to an inductive composition of immune responses against respiratory syncytial virus (RSV) that provides one or more RSV antigens, for use in a method of providing protective immunity against RSV or the protection of a disease caused by RSV in a baby, in which the procedure comprises administering to the baby at approximately 4 months of age or younger, the immune-inducing composition directed against RSV, in which said baby was a newborn of a woman to which was administered a composition that provides one or more RSV antigens that induced an immune response directed against RSV during the time said woman was pregnant with said baby, in which the one or more RSV antigens comprise a glycoprotein RSV F. Accordingly, the methods disclosed to provide protective immunity against RSV in a baby, comprise (a ) administer an inductive composition of a first immune response against RSV to a woman during pregnancy; and (b) administer a second inductive composition of an immune response directed against RSV to the baby born from pregnancy.

The disclosure also refers to procedures for protecting a baby from RSV disease, which include administering to a baby an inductive composition of an immune response directed against RSV, in which the baby was born from a woman to the which was administered an inductive composition of an immune response directed against RSV during the time in which the woman was pregnant with the baby.

In some aspects the inductive composition of an immune response directed against RSV administered to a woman during pregnancy reinforces the response of a neutralizing antibody in women. The neutralizing antibody response can be characterized by a 2-fold or more increase in the neutralizing titer. The neutralizing antibody response can be characterized by neutralizing antibodies. In some embodiments, neutralizing antibodies are independent of complement. In some embodiments, administration to a woman during pregnancy is carried out during the second or third trimester of pregnancy. In some embodiments, Administration to the baby born from pregnancy occurs immediately after birth, approximately 2 weeks after birth, approximately 4 weeks after birth, approximately 6 weeks after birth, approximately 2 months after birth, approximately 3 months after birth. from birth or about 4 months after birth.

In some embodiments, the first inductive composition of an immune response directed against RSV and the second inductive composition of an immune response directed against RSV are the same. The first inducing compositions of an immune response directed against RSV can each comprise a composition with a subunit of RSV, a nucleic acid, a particle of a viral replicon, a live attenuated virus, an inactivated viral particle, a recombinant viral vector, or a virus-like particle. Inductive compositions of an immune response directed against RSV each comprise one or more peptides or a composition of a virosome.

In some embodiments, the first inductive composition of an immune response directed against RSV is not equal to the second inductive composition of an immune response directed against RSV. The first inducing composition of an immune response directed against RSV may comprise a composition of a RSV subunit, a nucleic acid, or a particle of a viral replicon; and the second inducing composition of an immune response directed against RSV may comprise a composition of a RSV subunit, a nucleic acid, or a particle of a viral replicon; with the proviso that the first and second compositions that induce an immune response directed against RSV are not both a composition of a subunit of RSV, a nucleic acid or a particle of a viral replicon. The first inducing composition of an immune response directed against RSV may comprise a composition of a RSV subunit, a nucleic acid, a recombinant viral vector, or a particle of a viral replicon; and the second inducing composition of an immune response directed against RSV may comprise a composition of a RSV subunit, a nucleic acid, a recombinant viral vector, or a particle of a viral replicon; with the proviso that the first and second compositions that induce an immune response directed against RSV are not both a composition of a subunit of RSV, a nucleic acid or a particle of a viral replicon. In some embodiments, the first inductive composition of an immune response directed against RSV comprises a composition of a RSV subunit. In some embodiments, the second inducing composition of an immune response directed against RSV comprises a nucleic acid or a particle of a viral replicon. In some embodiments, the second inducing composition of an immune response directed against RSV comprises a nucleic acid, a recombinant viral vector, or a particle of a viral replicon.

In a preferred aspect, the first inductive composition of an immune response directed against RSV that is administered to a woman during pregnancy is not replicated in the woman after administration, and the second inductive composition of an immune response directed against RSV that It is given to the baby born from pregnancy does not replicate itself or its genome in the baby. For example, the first inducing composition of an immune response directed against RSV can be, for example, a composition of a RSV subunit, a particle similar to RSV virus (VLP), one or more RSV peptides, a composition of the mimitec epitope, a virosomal composition, or a composition of a destroyed RSV virion, and the second inducing composition of an immune response directed against RSV can be, for example, a self-replicating nucleic acid (e.g., a self-replicating (self-amplifying) RNA , a live attenuated virus, a heterologous live virus (for example a virus related to RSV with a non-primary human host), a live viral vector, a live chimeric virus, or a particle of a viral replicon.

Brief Description of the Figures

FIGS. 1A and 1B are graphs showing the correlation between serum neutralization titres and RSV stimulus protection in cotton rats (FIG. 1A) or BALB / c mice (FIG. 1B). Cotton rats and mice were injected ip with donor serum containing different levels of RSV-specific antibody (see the procedures of Example 3 and Tables 3 and 4 for more details) and stimulated in with RSV 2 days later . Each circle represents the serum neutralization titer one day after transfer and the viral load of the lung after stimulation (5 days after stimulation for cotton rats, 4 days after stimulation for mice) for an individual animal. The dashed lines indicate the limits of detection of the assay, and the solid line shows the curve that best fits 4 parameters for the data used to calculate the neutralization titer correlated with the virus not detectable after the stimulus.

Detailed description

As described herein, the inventors have discovered an immunization regime whereby a baby is protected against respiratory syncytial virus (RSV) by administering a first inductive composition of an immune response directed against RSV to this or to your mother (i.e., a pregnant woman) during pregnancy, followed by the administration of a second inductive composition of an immune response directed against RSV to the baby after birth. Immunization of the pregnant woman provides antibody-mediated immunity through passive maternal immunity. Passive immunity occurs naturally when maternal antibodies are transferred to the fetus through the placenta.

Passive immunity is especially important because babies are born without any actively acquired immunity. The administration of an inductive composition of an immune response directed against RSV to a pregnant woman enhances immunity to RSV in women, and antibodies directed against RSV are transmitted to the newborn through the placenta, conferring passive maternal immunity to baby. However, passive immunity in infants is only temporary and begins to decrease within a few weeks, or months of life. Since passive immunity is only temporary, it is important for the baby to receive the administration of an inductive composition of an immune response directed against RSV, to induce active immunity in the baby, before passive immunity decreases. The administration of a second immunogenic composition to the baby after birth induces active immunity against RSV in the baby, and extends the immunity transmitted by the mother during pregnancy.

The immunization regimen described herein provides some advantages. For example, this strategy provides protection from RSV infection to the baby from birth. An additional advantage is that administration of a composition of an immune response directed against RSV to the baby according to the regimen described herein may result in a greater induction of neutralizing RSV immunity than pre-immunization strategies. of the baby. The peak of serious illness due to RSV occurs at approximately two to three months of age. Immunization of pregnant women, which increases their antibody titres and passive antibody titres in their babies, could delay the onset of RSV disease in infants until a time when their respiratory systems are more mature and less likely to become seriously ill. The newborn's immune system is immature, making active immunization of newborns less effective than immunization of older babies. The delay of RSV disease in infants due to enhanced passive immunity from the immunization of the pregnant woman will allow more time for the active immunization of the baby before the age of greatest risk of serious illness. This added time for effective immunization could allow multiple doses of vaccines to be provided to a baby and / or provide doses later in childhood, when the immune system is more mature but even before the baby's first RSV infection or disease produced by the VRS.

As used herein, a baby is a one-year-old individual (for example, less than one day old, 1 week old, 2 weeks old, 3 weeks old, 4 weeks old, 2 months old, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months old, 9 months old, 10 months old, 11 months old, less than 12 months old).

As used herein, an inductive composition of an immune response directed against RSV is any composition that is suitable for administration to a mammal and is effective in inducing active immunity or boosting an immune response, preferably a immunoprotective response against RSV. The inducing composition of an immune response directed against RSV will provide one or more RSV antigens, and may be in the form of, for example, a composition of a subunit comprising a VRS protein antigen, a nucleic acid encoding a VRS protein antigen, a particle of a viral replicon (VRP) that contains a nucleic acid that encodes an antigen of a RSV protein, live attenuated viruses, inactivated viruses, virus-like particles (VLP), or recombinant viral vectors. The RSV antigen can be any desired RSV antigen. For example, the immune response inducing composition directed against RSV may comprise a RSV glycoprotein or a portion thereof (eg, ectodomain), preferably, a RSV F glycoprotein. RSV glycoprotein can be in any conformation ( for example, prefusion type conformation, post-fusion type conformation) or a mixture of conformations. The RSV antigen administered to the pregnant woman and the baby may be from different serogroups, different serotypes, different immunotypes, different serovars, different biovars, different strains, different clades, or different species and / or may contain nutations (e.g. deletions ). The RSV antigen administered to the pregnant woman and the baby can be of different subtypes.

The compositions inducing an immune response directed against RSV used in the immunization regimen may be the same or different. For example, a composition of a RSV subunit, a nucleic acid, a viral replication particle (VRP) or an attenuated virus during pregnancy can be administered to a woman and the same or different composition of the subunit of the baby is administered to the baby. RSV, a nucleic acid, a viral replication particle (VRP) or a virus attenuated after birth. In another example, an attenuated virus can be administered to the woman during pregnancy and a composition of a RSV subunit, a nucleic acid or a VRP is administered to the child after birth. In some cases, they are given to the baby and the pregnant woman attenuated vaccines. In other cases, the same or different inductive composition of an immune response directed against RSV selected from the group consisting of compositions of a subunit of RSV, nucleic acids, and VRP is administered to the baby and the pregnant woman. In other cases, a stimulus with an inductive composition of an immune response directed against RSV is administered to the baby and a reinforcement with another inductive composition of an immune response directed against RSV is administered. For example, the baby can be stimulated with a nucleic acid and reinforced with a subunit composition. In some cases, each of the baby or the pregnant woman or the baby and the pregnant woman is administered inductive compositions of an immune response directed against RSV simultaneously. For example, the baby, A nucleic acid and a subunit composition can be administered simultaneously to each of the pregnant woman, or the baby and the pregnant woman.

In some embodiments, the inductive composition of an immune response directed against RSV administered to women and the inductive composition of an immune response directed against RSV administered to the baby are not the same. For example, the inductive composition of an immune response directed against RSV administered to women may comprise a composition of a RSV subunit, a nucleic acid or a VRP, and the inductive composition of an immune response directed against RSV administered to the baby. it can comprise a composition of a subunit of RSV, a nucleic acid or a VRP, with the proviso that the compositions that induce an immune response directed against RSV administered to women and the baby are not both a composition of a subunit of RSV , a nucleic acid or a virus replication particle.

The inductive composition of an immune response against RSV administered to women will most likely be a booster. For example, the woman will probably have experienced an RSV infection at some point in her life before pregnancy, so that the inductive composition of an immune response directed against RSV administered to her during pregnancy will act as a stimulus. The booster will increase the antibody titer directed against RSV in the mother and will enhance the maternal immunity that is transmitted to the fetus and the baby.

The inductive composition of an immune response directed against RSV can be administered to the pregnant woman at any time during her pregnancy. For example, the inductive composition of an immune response directed against RSV can be administered to women during the first, second or third trimester of pregnancy. In some embodiments, the inductive composition of an immune response directed against RSV is administered to the woman during the last 6-12 weeks of pregnancy (eg, 28 weeks gestation, 29 weeks gestation, 30 weeks gestation, 31 weeks gestation, 32 weeks gestation, 33 weeks gestation, 34 weeks gestation, 35 weeks gestation, 36 weeks gestation, 37 weeks gestation, 38 weeks gestation, 39 weeks gestation). Preferably, the inductive composition of an immune response directed against RSV is administered to the pregnant woman for at least four weeks before administration to the baby.

In some embodiments, a one-dose regimen is administered to the pregnant woman between 32 and 36 gestation.

In other embodiments, a two-dose regimen is administered to the pregnant woman, the first dose being administered at approximately 32 weeks gestation and the second dose administered at approximately 36 weeks gestation.

An inductive composition of an immune response directed against RSV can be administered to the baby at any time during the first year of life, and subsequently if desired. Generally, an inductive composition of an immune response directed against RSV will be administered to the baby one, two, three, four or more times during the first year of life, and the compositions may be the same or different. For example, an inductive composition of an immune response directed against RSV can be administered to the baby one or more times selected from birth, at 2 weeks of age, 4 weeks of age, 6 weeks of age, 2 months of age , 3 months old, 4 months old, 6 months old, 9 months old, and 12 months old. Preferably, the inductive composition of the immune response directed against RSV is administered to the baby at a time before the maternal antibodies to non-protective titers have decreased, therefore, in accordance with the present invention, the inductive composition of a response Immune directed against RSV is initially given to the baby at approximately 4 months of age or younger. Subsequent administrations can occur on any desired calendar.

For example, an inductive composition of an immune response directed against RSV can be administered to the baby as a three dose regimen. A possible dosing regimen includes administration at the time of birth, 1 month of age, and 2 months of age. Another possible dosage regimen includes administration at 1 month of age, 2 months of age and 3 months of age. Another illustrative dosage regimen includes administration at 1 month of age, 2 months of age and 4 months of age.

The administration of the inducing composition of an immune response directed against RSV can be at practically the same time as other vaccines (for example, during the same consultation or medical visit to a healthcare professional or to a vaccination center) or at a later time initial given, at the beginning of the onset of RSV disease.

In some embodiments, the inductive composition that induces the immune response directed against RSV comprises proteins, DNA molecules, self-replicating RNA molecules or VRP. The inductive composition of an immune response directed against RSV may further comprise a pharmaceutically acceptable carrier and, optionally, an adjuvant. See, for example, US 6,299,884; the US document

7,641,911; the U.S. document 7,306,805; and US 2007/0207090. Certain inductive compositions of an immune response directed against RSV are further described herein.

The immune response may comprise a humoral immune response, a cell-mediated immune response, or both. In some embodiments an immune response is induced against each RSV protein administered. A cell-mediated immune response may comprise an auxiliary T (T) lymphocyte response, a CD8 + cytotoxic T lymphocyte (LTC) response, or both. In some embodiments, the immune response comprises a humoral immune response and the antibodies are neutralizing antibodies. Neutralizing antibodies block viral infection of the cells. In some embodiments, the immune response reduces or prevents infection of the cells. Neutralizing antibody responses may be complement dependent or complement independent. In some embodiments, the neutralizing antibody response is independent of complement. In some embodiments, the neutralizing antibody response is cross neutralization; that is, an antibody generated against a administered composition neutralizes a VRS virus from a different strain of the strain used in the composition. In some embodiments, the neutralizing antibody response is characterized by neutralizing IgG antibodies.

A useful measure of antibody potency in the art is "% neutralization of the title" (eg, 50% neutralization of the title, 60% neutralization of the title). To determine the% neutralization of the titer, the serum of the immunized individuals is diluted to assess how the dilution of the serum can also retain the ability to block the entry of 50% or 60%, respectively, of infectious viruses into the cells. For example, a 50% neutralization of a titer of 20 means that the serum retains the ability to neutralize 50% of the virus after being diluted 20 times. Therefore, larger titers indicate more potent neutralizing antibody responses. In some embodiments, this title is in a range that has a lower limit of about 20, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or about 200. The variation of the neutralization title of the 50% may have an upper limit of about 40, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 400, about 800, about 1600, about 3000, about 6000, about 8,000, about 10,000 or about 20,000. For example, the 50% neutralization titer may be about 1:20, 1:40, 1:80, or about 1: 100. "Approximately" means twice the value listed. The neutralization titer can be measured as described herein.

The neutralization titer can be measured by mixing an RSV preparation with an antibody preparation, adding the mixture to cultured mammalian cells and then determining the infectivity ratio of the original RSV that remains in the antibody-virus mixture evaluating the consequences of adding the Mix the cells. The preparation containing the antibody may include, for example, animal serum, human serum, antibodies purified from serum, or monoclonal antibodies from culture. In some cases the complement, for example, the guinea pig complement, can be mixed with the antibody and the RSV preparation. In some embodiments, serial dilutions of the preparation containing the antibody are added to the virus samples. The mammalian cells used in the neutralization assay may be in a monolayer or in suspension, and may be infected. The inoculum, which can include viruses and an antibody, can remain in the cells or can be removed after a short time (for example, 30 minutes, 60 minutes, or 90 minutes). The proportion of infected cells can be determined by a variety of means, including, for example, counting plates that form a semi-solid coating layer, counting syncytiums that form a semi-solid coating layer, immunostained infectious foci after a brief incubation with a virus , detection of a fluorescent marker expressed in a recombinant RSV, identification of infected cells observing the cytopathic effect, identification of infected cells by immunostaining of a RSV marker, evaluate the amount of RSV antigen in the supernatant of intact or lysed cells, detect the amount of fluorescence of a recombinant RSV that expresses a fluorescent antigen, detect the amount of cell lysis by a biochemical assay (eg, LDH), and observe the highest dilution of virus at which any cytopathic effect is observed after a while of sufficient incubation. Additional variations in the neutralization assay may include, for example, variations in buffers, blocking agents, incubation times, controls, plate type, incubation temperatures, cell culture medium, permeabilizing agent, fixing agent, type of Cell substrate, coating composition, cell staining, reagent volume, number of washes, use of secondary antibodies, counting procedure, calculation procedure, and neutralization percentage used to calculate the titer.

An immune response can be stimulated by administering an inductive composition of an immune response directed against RSV (a composition comprising proteins, DNA molecules, self-replicating RNA molecules or VRP) to an individual. In some embodiments, the induced immune response is an immunoprotective response, that is, the response reduces the risk of RSV infection, delays the onset of RSV infection, reduces the risk of RSV disease, reduced the severity of the disease produced. by VRS, delays the onset of RSV disease reduces the frequency of RSV disease, reduces the sequelae of RSV infection or RSV disease, or reduces the risk that an individual infected with RSV will spread RSV to another individual.

Any suitable administration route can be used. For example, a composition can be administered intramuscularly, intraperitoneally, subcutaneously, transdermally or intradermally. If desired, the composition may be administered through an intramucosal route such as by an intraoral, intranasal, intravaginal, and intrarectal route. administration to the pregnant woman and the baby can be through the same route or different routes. The compositions can be administered according to some suitable calendar.

As described herein, a protective immunity against RSV is provided to a baby comprising the steps of (a) administering a first inductive composition directed against RSV to the woman during pregnancy; and (b) administer a second inductive composition of an immune response directed against RSV to the baby born from pregnancy. As described herein, the first inductive composition of an immune response directed against RSV and the second inductive composition of an immune response directed against RSV may be the same or different. This strategy preferably induces or reinforces the neutralization of antibodies directed against RSV in pregnant women that are transmitted to the baby through maternal immunity, and induces an active immune response in the baby. Therefore, preferably, at the time of the first administration of the inductive composition of an immune response directed against RSV to the baby, the baby contains maternal antibodies against RSV, and as a result of the administration will actively develop an immune response against the VRS Preferably, the composition of an immune response directed against RSV is administered to the baby at a time when the maternal antibodies are still at protective levels in the baby.

In a particular embodiment, protective immunity against RSV is provided in a baby by administering a composition of a subunit directed against RSV to a woman during pregnancy; and administering a self-replicating RNA composition directed against RSV to a baby born from pregnancy. In a preferred embodiment, protective immunity against RSV is provided in a baby by administering a composition of a subunit directed against RSV to a woman during pregnancy; and administering one or more self-replicating RNA compositions directed against RSV to a baby born from pregnancy, followed by administering a reinforcement composition of the subunit directed against RSV to the baby.

As described herein, a baby is protected from infection caused by RSV by a procedure comprising administering to a baby an inductive composition of an immune response directed against RSV, in which the baby was born from a woman to which was given a booster or inducer composition of an immune response directed against RSV during the time the woman was pregnant with the baby. The inductive composition of an immune response directed against RSV that was administered to the woman and the inductive composition of an immune response directed against RSV that was administered to the baby may be the same or different. Preferably, at the time of the first administration of the inducing composition of an immune response directed against RSV to the baby, the baby contains maternal antibodies against RSV, and as a result of administration it will actively develop an immune response against RSV. Preferably, the inductive composition of an immune response directed against RSV is administered to the baby at a time when maternal antibodies are still in protective titers in the baby.

In a preferred embodiment, a baby is protected from RSV infection by administering a self-replicating RNA composition directed against RSV to a baby born to a woman to whom a subunit composition directed against RSV was administered for as long as the Woman was pregnant with the baby.

Inductive composition of an immune response directed against RSV

Any inductive composition of an immune response directed against the desired RSV can be used. For example, RSV proteins or fragments can be administered directly as components of an inductive composition of an immune response directed against RSV, or nucleic acids encoding one or more proteins or fragments can be administered to produce the RSV protein or fragment in. live, or live attenuated viruses can be administered, or recombinant viral vectors expressing RSV proteins can be administered, or RSV viruses can be administered, or virus-like particles can be administered containing viruses containing RSV antigens. In certain preferred embodiments, such as protein formulations, recombinant nucleic acids (eg, self-replicating RNA) and VRP alphavirus containing sequences encoding proteins or fragments of v Rs are further described herein.

Protein formulations

Proteins or immunogenic fragments thereof used in accordance with the invention will normally be isolated or purified. Therefore, they will separate and differentiate from the whole organism or virion with which the molecule is found in nature or is present in the substantial absence of other biological macromolecules of the same type.

RSV exists as a single serotype, but it has two antigenic subgroups: A and B. The F glycoproteins of the two Groups are approximately 90 % identical. Subgroup A, subgroup B, or a combination or hybrid of both may be used in the invention. An illustrative sequence for subgroup A is SEQ ID NO: 1 (strain A2; Gi of GenBank: 138251; Swiss Prot P03420), and for subgroup B is SEQ ID NO: 2 (strain 18537; GI: 138250; Swiss Prot P13843). The s Eq ID NO: 1 and SEQ ID NO: 2 have sequences of 574 amino acids. The signaling peptide in strain A2 has aa 1-21, but in strain 18537 it has aa 1-22. In both sequences the TM domain has approximately aa 530-550, but has been reported alternatively as 525-548.

SEQ ID NO: 1

1 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIE 60

61 LSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLN 120

121 NAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAWS 180

181 LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVN 240

241 AGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV 300

301 VQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV 360

361 QSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKT 420

421 KCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP 480

481 LVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLS 540

541 LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN 574

SEQ ID NO: 2

1 MELL1HRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIE 60

61 LSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTIN 120

121 TTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAWS 180

181 LSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVN 240

241 AGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV 300

301 VQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKV 360

361 QSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKT 420

421 KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDP 480

481 LVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIWLLS 540

541 LIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK 574

The RSV proteins may contain one or more mutations that prevent cleavage at one or both of the furin cleavage sites (ie amino acids 109 and 136 of SEQ ID NOS: 1 and 2). These mutations can prevent the aggregation of soluble polypeptides or proteins and therefore facilitate purifications, can prevent cell-cell fusion if the RSV F protein is expressed on the surface of a cell, such as by the expression of a viral replicon ( alphavirus replicon particles), or if the VRS F protein is a component of a virus-like particle. These mutations, alone or in combination with other mutations described herein, can also stabilize the protein in the prefusion type conformation.

Examples of suitable furin cleavage mutations include the replacement of amino acid residues 106-109 of SEQ ID NO: 1 or 2 with RARK (SEQ ID NO: 3), RARQ (SEQ ID NO: 4), QAQN ( SEQ ID NO: 5), or IEGR (SEQ ID NO: 6). Alternatively or in addition, amino acid residues 133-136 of SEQ ID NO: 1 or 2 may be substituted with RKKK (SEQ ID NO: 7), ® ® ® R, QNQN (SEQ ID NO: 8), QQQR (SEQ ID NO: 9) or IEGR (SEQ ID NO: 10). (® indicates that the remaining amino acid has been removed.) These mutations can be combined, if desired, with other mutations described herein, such as mutations in the p27 region (amino acids 110-136 of SEQ ID NOS: 1 or 2), including the deletion of the p27 region in whole or in part.

These furin cleavage mutations, if desired, can be combined with other mutations described herein, such as trypsin cleavage mutations and fusion peptide mutations. Examples of suitable trypsin cleavage mutations include the deletion of any lysine or arginine moiety between about position 101 and position 161 of SEQ ID NO: 1 or 2 or the substitution of any such lysine or arginine moiety. with an amino acid different from lysine or arginine. For example, the lysine and / or arginine residues in the p27 region (approximately amino acids 110-136 of SEQ ID NOS: 1 or 2) can be substituted or removed, including the deletion of the whole or in part p27 region.

As an alternative or in addition to furin cleavage mutations, RSV F polypeptides or proteins may contain one or more mutations in the fusion peptide region (amino acids 137 and 153 of SEQ ID NOS: 1 or 2). For example, this region can be removed completely or in part.

In particular embodiments, the sequence of the amino acid residue 100-150 of the VRS protein, such as SEQ ID NO: 1, SEQ ID NO: 2, or the soluble ectodomains thereof, are

(Deletion 1 of the fusion peptide)

TPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRR ----- SAIAS (SEQ ID NO: 11), or

(Deletion 2 of the fusion peptide)

TPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRR ----- GVGSAIAS (SEQ ID NO: 12).

In addition to furin cleavage mutations and fusion peptide mutations, or, alternatively, soluble RSV proteins, such as those lacking the transmembrane region and the cytoplasmic tail, RSV proteins may contain one or more. more oligomerization sequences. When an oligomerization sequence is present, it is preferably a trimerization sequence. Suitable oligomerization sequences are well known in the art and include, for example, the coiled spiral of the yeast GCN4 zipper protein, The trimerization sequence of the bacteriophage T4 ("foldon" fibritin) and the trimeric domain of para-influenza HA These and other suitable oligomerization sequences are described in more detail herein.

The proteins or fragments thereof, will usually be prepared by expression in a recombinant host system. In general, they are produced (for example, VRS proteins) by the expression of recombinant constructs encoding the proteins in suitable recombinant host cells, although any suitable methods can be used. Suitable recombinant host cells include, for example, insect cells (e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni), mammalian cells (e.g., human, nonhuman primates , horse, cow, sheep, dog, cat, and rodent (e.g., hamster), bird cells (e.g., chickens, ducks, and geese), bacteria (e.g., E. coli, Bacillus subtilis, and Streptococcus spp .), yeast cells (for example, Saccharomyces cerevisiae, Candida albicans, Candida maltose, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia teolytramena (Temples thermotramena), for example ) or combinations thereof. Many suitable insect cells and mammalian cells are well known in the art. Suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a cloned isolate derived from the BTI-TN-5B1-4 precursor line of Trichoplusia ni (Invitrogen)). Suitable mammalian cells include, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 cells, normally transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293- cells. T, Vero cells, HeLa cells, PERC.6 cells (ECACC deposit number 96022940), Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), fetus lung cells of rhesus (ATCC CL-160), Madin-Darby bovine kidney cells ("MDBK"), Madin-Darby canine kidney cells ("MDCK") (for example, Md CK (NBL2), ATCC CCL34; or MDCK 33016, DSM ACC 2219), hamster breeding kidney (BHK) cells, such as BHK21-F, HKCC cells, and the like. Suitable poultry cells include, for example, chicken embryocytoblasts (eg, EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, duck cells (for example AGE1.CR and AGE1.CR cell lines. pIX cell lines (ProBioGen) described, for example, in Vaccine 27: 4975-4982 (2009) and WO2005 / 042728), EB66 cells, and the like.

Those skilled in the art are aware of suitable insect cell expression systems, such as baculovirus systems, and are described in, for example, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and procedures for baculovirus / insect cell expression systems are commercially available in kit form, among others, Invitrogen, San Diego CA. Bird cell expression systems are also known to those skilled in the art and are described in, for example, US Pat. Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; European Patent No. EP 0787180B; European Patent Application No. EP03291813.8; WO 03/043415; and WO 03/076601. Similarly, bacterial and mammalian cell expression systems are also known in the art and described, for example, Yeast Genetic Engineering (Barr et al., Eds., 1989) Butterworths, London.

Recombinant constructs encoding RSV proteins can be prepared in suitable vectors using conventional procedures. Numerous vectors suitable for the expression of recombinant proteins in insect or mammalian cells are well known and conventional in the art. Suitable vectors may contain numerous components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcription control element (eg, a promoter, an enhancer, a terminator), and / or one or more translation signals; and a signaling sequence or a leader sequence for targeting the secretory pathway in a selected host cell (for example of mammalian origin, or of a heterologous mammalian or non-mammalian species). For example, for expression in insect cells of a suitable baculovirus expression vector, such as pFastBac (Invitrogen), it is used to produce recombinant baculovirus particles. Baculovirus particles are amplified and used to infect insect cells that express the recombinant protein. For expression in mammalian cells, a vector is used that will drive the expression of the construct in the host cell of the desired mammal (eg, Chinese hamster ovary cells).

The proteins can be purified using any suitable procedures. For example, methods for purifying RSV proteins by immunoaffinity chromatography are known in the art. Ruiz-Arguello et al., J. Gen. Virol., 85: 3677-3687 (2004). Suitable procedures for purifying desired proteins that include precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelation and molecular exclusion are well known in the art. Suitable purification schemes can be created using two or more of these or other suitable procedures. If desired, RSV proteins may include a "tag" that facilitates purification, such as an epitope tag or an HIS tag. Said labeled proteins can be conveniently purified, for example, from conditioned media, by chelation chromatography or affinity chromatography. Additional purification techniques include diafiltration and filtration in tangential flow.

Proteins may include additional sequences in addition to the RSV sequences. For example, a polypeptide may include a sequence to facilitate purification (for example, a poly-His sequence with or without a linker). Similarly, for the purpose of expression, the natural leader peptide may be substituted for a different one.

In some embodiments, RSV proteins are administered using alphavirus replicon particles (VRP). Any nucleotide sequence encoding a RSV protein can be used to produce the protein. As used herein, the term "alphavirus" has its conventional meaning in the art and includes various species such as the equine encephalitis virus of Venezuela (VEE; for example, Donkey of Trinidad, TC83CR, V3014 etc. ), Semliki Forest Virus (SFV), Sindbis Virus, Ross River Virus, Western Equine Encephalitis Virus, Eastern Equine Encephalitis Virus, Chikungunya Virus, SA AR86 Virus, Everglades Virus, Mucambo Virus, Barmah Forest Virus, Virus Middelburg, Pixuna Virus, O'nyong-nyong Virus, Getah Virus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, Banbanki Virus, Kyzylagach Virus, Highlands J Virus, Fort Morgan Virus, Ndumu virus, and Buggy Creek virus virus.

An "alphavirus replicon particle" (VRP) or a "replicon particle" is an alphavirus replicon packaged with alphavirus structural proteins.

An "alphavirus replicon" (or "replicon") is an RNA molecule that can direct its own amplification in vivo in a target cell. Replicon encodes the polymerase (s) that catalyzes RNA amplification (nsP1, nsP2, nsP3, nsP4) and contains cis RNA sequences required for replication that are recognized and used by the polymerase (s) ) encoded (s). an alphavirus replicon normally contains the following ordered elements: 5 'viral sequences required for cis replication, sequences encoding biologically active alphavirus nonstructural proteins (nsP1, nsP2, nsP3, nsP4), 3' viral sequences required for cis replication, and an extension of polyadenylate. An alphavirus replicon may also contain one or more promoters of a viral subgenomic "binding region" that direct the expression of heterologous nucleotide sequences, which may, in certain embodiments, be modified in order to increase or reduce the viral transcription of the fragment. subgenomic and the sequence (s) to be expressed. Other elements of the control can be used, as described below.

Alphavirus replicons encoding one or more RSV proteins are used to produce VRP. Said alphavirus replicons comprise sequences encoding one or more RSV proteins or fragments thereof. These sequences are operably linked to one or more suitable control elements, such as a subgenomic promoter, an IRES (eg, VEMC, EV71), and a viral 2A site, which may be the same or different. Any one or a combination of suitable control elements can be used in any order.

The use of polycistronic vectors is an efficient way of providing nucleic acid sequences encoding two or more RSV proteins in desired relative amounts. In one example, a single subgenomic promoter is operatively linked to two sequences encoding two different RSV proteins, and an IRES is placed between the two coding sequences. In another example, two sequences encoding two different RSV proteins are operatively linked to separate promoters. In another example, the two sequences encoding two different RSV proteins are operatively linked to a single promoter. The two sequences encoding two different RVS proteins bind to each other through a nucleotide sequence that encodes a viral 2A site, and thus encodes a single amino acid chain that contains the amino acid sequences of both RSV proteins. The viral 2A site in this context is used to generate two RSV proteins from the original polyprotein.

Subgenomic promoters

Subgenomic promoters, also known as promoters of the binding region can be used to regulate protein expression. Alpha-viral subgenomic promoters regulate the expression of alpha-viral structural proteins. see Strauss and Strauss, "The alphaviruses: gene expression, replication, and evolution", Microbiol Rev. sep. 1994; 58 (3): 491-562. A polynucleotide may comprise a subgenomic promoter from any alphavirus. When two or more subgenomic promoters are present, for example, in a polycistronic polynucleotide, the promoters may be the same or different. For example, the subgenomic promoter may have the sequence CTCTCTACGGCTAACCTGAATGGA (SEQ ID NO: 13). In certain embodiments, the subgenomic promoters may be modified in order to increase or reduce the viral transcription of the proteins. See U.S. Patent No. 6,592,874.

Internal ribosome entry site (IRES)

In some embodiments, one or more control elements is an internal ribosome entry site (IRES). An IRES It allows multiple proteins to be prepared from a single mRNA transcript such as ribosomes that bind to each IRES and initiate translation in the absence of a 5 'cap, which is normally required to initiate translation. For example, IRES can be obtained from the cloned genome of enterovirus 71 (EV71) or encephalomyocarditis virus (VEMC).

Viral site 2A

The 2A protein of the foot and mouth disease virus (FMDV) is a short peptide that serves to separate the structural proteins of the FMDV from a non-structural protein (FMDV 2B). Initial work on this peptide suggested that it acts as an autocatalytic protease, but other work (for example, Donnelly et al. (2001), J.Gen.Virol. 82, 1013-1025) suggests that this sequence cuts and follows Individual amino acid of FMDV 2B (Gly) acts as a stop-start of translation. With respect to the precise mode of action, the sequence between two polypeptides can be inserted, and multiple individual polypeptides can be produced from a single open reading frame. The FMDV 2A sequences can be inserted between the sequences encoding at least two RSV proteins, allowing their synthesis as part of a single open reading frame. For example, the open reading frame can encode an RL11 protein and an UL119 protein separated by a sequence encoding a viral 2A site. A single mRNA is then transcribed, during the translation stage, the RL11 and UL119 peptides are produced separately due to the activity of the viral 2A site. Any viral 2A sequence can be used. Often, a viral 2A site comprises the Asp-Val / Ile-Glu-X-Asn-Pro-Gly-Pro consensus sequence, in which X is an amino acid (SEQ ID NO: 14). For example, the peptide 2a sequence of the foot and mouth disease virus is DVESNPGP (SEQ ID NO: 15). See Trichas et al., "Use of the viral 2A peptide for bicistronic expression in transgenic mice", BMC Biol. 15 Sep 2008 15; 6: 40, and Halpin et al., "Self-processing 2A-polyproteins - a system for co-ordinate expression of multiple proteins in transgenic plants ", Plant J. Feb 1999; 17 (4): 453-9.

In some embodiments, an alphavirus replicon is a chimeric replicon, such as a VEE-Sindbis chimeric replicon (VCR), a TC83 replicon of the VEE strain (TC83R), or a TC83-Sindbis chimeric replicon (TC83CR), or a V3014 replicon of the VEE strain. In some embodiments, a VCR contains the packaging signal and the 3 'UTR of a Sindbis replicon instead of the sequences in nsP3 and at the 3' end of the VEE replicon; see Perri et al., J. Virol. 77, 10394-403, 2003. In some embodiments, a TC83CR contains the packaging signal and the 3 'UTR of a Sindbis replicon instead of sequences in nsP3 and at the 3' end of a TC83 replicon of a VEE strain.

VRP production

Methods of preparing VRP are well known in the art. In some embodiments. an alphavirus is assembled in a VRP using a packed cell. An "alphavirus packed cell" (or "packed cell") is a cell that contains one or more alphavirus structural protein expression cassettes and produces recombinant alphavirus particles after the introduction of an alphavirus replicon, the starting system stratified from a eukaryotic vector, for example, U.S. Patent 5,814,482), or a recombinant alphavirus particle. The one or more different alphavirus structural protein cassettes serve as "auxiliaries" by providing the alphavirus structural proteins. An "alphavirus structural protein cassette" is an expression cassette that encodes one or more alphavirus structural proteins and comprises at least one and up to five copies (ie, 1, 2, 3, 4, or 5) of a sequence of recognition of an alphavirus replicase. Structural protein expression cassettes typically comprise, from 5 'to 3', a 5 'sequence that initiates transcription of the alphavirus RNA, an optional promoter of the subgenomic region of alphavirus, a nucleotide sequence that encodes the structural protein of the alphavirus, a 3 'untranslated region (which also directs RNA transcription), and a polyA extension. See, for example, WO 2010/019437.

In one embodiment, two different alphavirus structural protein cassettes ("split" defective auxiliaries are used in a cell package to minimize recombination episodes that could produce a competent virus for replication. In some embodiments a structural protein cassette of alphavirus encodes the protein of capsid (C) but not any of the glycoproteins (E2 and E1) In some embodiments, an alphavirus structural protein cassette encodes the protein of the capsid and any of the E1 or E2 glycoproteins (but not both) In some embodiments, an alphavirus structural protein structural protein cassette encodes the E2 and E1 glycoproteins, but not the capsid protein In some embodiments, an alphavirus structural protein cassette encodes the E1 or E2 glycoprotein ( but not both) and not the capsid protein.

In some embodiments, VRPs are produced by the simultaneous introduction of replicons and helper RNAs into cells from various sources. Under these conditions, for example, BHKV or Vero cells (1x107) are electroporated in, for example, 220 volts, 1000 jF, 2 manual pulses with 10 | jg of replicon RNA: 6 | jg with auxiliary RNA with defective cap: 10 jg of defective auxiliary Gly RNA, Supernatant or cells containing VRP with associated RSVs are collected ~ 24 hours later. Replicons and / or auxiliaries can also be introduced into DNA forms that initiate suitable RNAs into the transfected cells.

A packaged cell can be a mammalian cell or a non-mammalian cell, such as an insect cell (e.g. SF9) or a bird cell (e.g., a larger chick or a duck fibroblast or a cell line of fibroblast). See U.S. Patent 7,445,924. Bird cell sources include, although not limitingly, bird embryoblasts such as EB66® (VIVALIS); chicken cells, including chicken embryoblasts, such as EBx® cells, chicken embryofibroblasts, and chicken embryonic germ cells; Duck cells (for example AGE1.CR and AGE1.CR.pIX (ProBioGen) cell lines which are described, for example, in Vaccine 27: 4975-4982 (2009) and WO2005 / 042728; and goose cells. In some embodiments, a packed cell is a primary duck fibroblast or a duck retinal cell line, such as AGE.CR (PROBIOGEN).

Sources of mammalian cells for the simultaneous introduction of nucleic acid and / or packaged cells include, but are not limited to, human or non-human primate cells, including PerC6 (PER.C6) cells (Cr Uc ELL NV), which they are described, for example, in WO 01/38362 and WO 02/40665, as well as those deposited with the ECACC deposit number 96022940; MRC-5 (ATCC CCL-171); WI-38 (ATCC CCL-75); macaque fetus lung cells (ATCC CL-160); human embryo kidney cells (for example, 293 cells, normally transformed by sheared adenovirus type 5 DNA); VERO monkey kidney cells; horse, cow (e.g., MDBK cells), sheep, dog cells (e.g., MDCK cells of dog kidneys, ATCC CCL34 MDCK (n Bl2) or MDCK 33016, deposit number Ds M Ac C 2219 as described in WO 97/37001); cat, and rodent (for example, hamster cells such as BHK21-F, HKCC cells, Chinese hamster ovary (CHO) cells), and can be obtained from a wide variety of developmental stages, including, for example, adult , newborn, fetus, and embryo.

In some embodiments, a packed cell is stably transformed with one or more structural protein expression cassettes. Structural protein expression cassettes can be introduced into cells using standardized recombinant DNA techniques, including transferring DNA mediated by transferrin polycation, mediated transfection with pure or encapsulated nucleic acids, cell fusion mediated by liposome, intracellular transport of coated latex beads of DNA, protoplast fusion, viral infection, electroporation, "gene gun" procedures, and transfection mediated by DEAE or calcium phosphate. Structural protein expression cassettes are normally introduced into a host cell as DNA molecules, but can also be introduced as RNA transcribed in vitro. Each expression cassette can be introduced separately or virtually simultaneously.

In some embodiments, stable alphavirus packed cell lines are used to produce recombinant alphavirus particles. These are permissive cells for the alphavirus comprising DNA cassettes that express the defective auxiliary RNA stably integrated into their genomes. See Polo et al., Proc. Natl Acad. Sci. USA 96, 4598-603, 1999. For example, in one embodiment the auxiliary RNAs are constitutively expressed but the structural proteins of the alphavirus do not, because the genes are under the control of a subgenomic alphavirus promoter (Polo et al., 1999). In another embodiment, the subgenomic promoter is not used in auxiliary RNAs to boost structural protein expression. After the introduction of an alphavirus replicon into the genome of a packed cell through VRP transfection or infection, replicase enzymes are produced and stimulate the expression of the capsid and glycoprotein genes in the auxiliary RNAs, and the output occurs of VRP. The introduction of the replicon can be carried out by a variety of procedures, including transfection and infection with a stock solution of seeding alphavirus replicon particles. The packed cell is then incubated under conditions and for a sufficient time to produce packaged particles of alphavirus replicon in the culture supernatant.

Therefore, packaged cells allow VRPs to act as self-propagating viruses. This technology allows VRPs to be produced in the same way, and use the same equipment, such as that used for live attenuated vaccines or other viral vectors that have available producer cell lines, said adenoviral vectors incompetent for replication grow in cells that express adenovirus genes E1A and E1B.

In some embodiments, a two-stage procedure is used: the first step comprises producing a stock solution of sowing alphavirus replicon particles by transfecting a packed cell with a plasmid DNA based replicon. A much larger stock solution of replicon particles is then produced in a second stage, infecting a recent culture of packed cells with the stock planting solution. This infection can be carried out using various multiplicities of infection (MOI), including an MOI = 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1 , 0.5, 1.0, 3, 5, 10 or 20. In some embodiments, infection is carried out at a low MOI (for example, less than 1). Over time, replicon particles can be collected from packaged cells infected with the stock planting solution. In some embodiments, the replicon particles can then be transferred to even larger cultures of packaged cells not previously treated by repeated low multiplicity infection, resulting in preparations on a commercial scale with the same high titer.

Nucleic acid compositions

Recombinant nucleic acid molecules encoding one or more RSV proteins or fragments can be administered to induce the production of encoded RSV proteins or fragments and an immune response thereto. The recombinant nucleic acid may be based on any desired nucleic acid such as DNA (for example, plasmid or viral DNA) or RNA, preferably, the self-replicating RNA, and may be monocistronic or polycistronic. Any suitable DNA or RNA can be used as the nucleic acid vector that carries the Open reading frames that encode RSV proteins or fragments thereof. Suitable nucleic acid vectors have the ability to transport and boost the expression of one or more RSV proteins or fragments. Such nucleic acid vectors are known in the art and include, for example, plasmids, DNA obtained from DNA viruses such as vaccinia virus vectors (eg, NYVAC, see US 5,494,807), adenovirus vectors, and vector vectors. of the smallpox virus (eg, vector of the ALVAC canarypox virus, Sanofi Pasteur), and RNA obtained from suitable RNA viruses such as alphavirus. If desired, the recombinant nucleic acid molecule can be modified, for example, to contain modified nucleobases and / or bonds, as further described herein.

Recombinant nucleic acid molecules that are polycistronic provide the advantage of releasing sequences encoding two or more RSV proteins to a cell, and for example, boosting the expression of RSV proteins to levels sufficient to result in the formation of a complex. of proteins containing the two or more RSV proteins in vivo. Using this strategy, two or more encoded RSV proteins that form a complex can be expressed at intracellular levels sufficient for the formation of RSV protein complexes. For example, encoded RSV proteins or fragments thereof can be expressed at virtually the same level, or, if desired, at different levels by selecting the appropriate expression control sequences (eg, promoters, IRES, site 2A , etc.). This is a significantly more efficient way to produce protein complexes in vivo than to co-administer two or more individual DNA molecules encoding different RSVs in the same cell, which can be inefficient and highly variable. See, for example, WO 2004/076645.

The "self-replicating" nucleic acids described herein are also referred to by the inventors and herein as "self-amplifiers." The terms self-replicating and self-amplifying are intended to have the same meaning.

The self-replicating RNA molecules of the invention are based on the genomic RNA of the RNA viruses, but lack the genes encoding one or more structural proteins. Self-replicating RNA molecules are capable of being translated to produce non-structural RNA virus proteins and RSV proteins encoded by the self-replicating RNA.

The self-replicating RNA generally contains at least one or more genes selected from the group consisting of viral replicase, viral proteases, viral helicases and other non-structural viral proteins, and also comprises cis-active replication sequences at the 5 'and 3 ends. ', and heterologous sequences encoding one or more desired RSV proteins. A subgenomic promoter that directs the expression of heterologous sequences in the self-replicating RNA can be included. If desired, a heterologous sequence can be fused in frame with other coding regions in the self-replicating Rn AM-vac that may be under the control of an internal ribosome entry site (IRES).

The self-replicating RNA molecules of the invention can be designed such that the self-replicating RNA molecule cannot induce the production of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary for the production of viral particles in the self-replicating RNA. For example, when the self-replicating RNA molecule is based on an alphavirus, such as Sindbis virus (SIN), Semliki forest virus and Venezuelan equine encephalitis virus (VEE), one or more genes encoding proteins can be omitted viral structures, such as capsid proteins and / or the envelope. If desired, the self-replicating RNA molecules of the invention can be designed to induce the production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single subsequent infection cycle.

A self-replicating RNA molecule can, when supplied to a vertebrate cell, even without any protein, lead to the production of multiple RNAs derived by transcription thereof (or a copy of the opposite direction thereof). The self-replicating RNA can be translated directly after administration to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces transcripts of the administered RNA. Therefore, the supplied RNA leads to the production of multiple derived RNAs. These transcripts are in the opposite direction with respect to the administered RNA and can be translated by themselves to provide in situ expression of the encoded RSV protein, or can be transcribed to provide additional transcripts in the same sense as the administered RNA that is translated to provide in situ expression of encoded RSV proteins.

A suitable system for achieving self-replication is to use an alpha-virus-based RNA replicon, such as the alpha-virus replicon described herein. These strand replicons are then translated administration to a cell to give rise to a replicase (or replicase transcriptase). Replicase is translated as a polyprotein that cleaves itself to provide a replication complex that creates copies of genomic strands of the RNA administered to the strand. These strand transcripts - can transcribe themselves to give additional copies of the strand precursor RNA and also to give a subgenomic transcript encoding two or more RSV proteins. Translation of the subgenomic transcript thus leads to in situ expression of RSV proteins by the infected cell. Suitable alphavirus replicons can use a replicase of a sindbis virus, a semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc.

A preferred self-replicating RNA molecule thus encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule and (ii) one or more RSV proteins or fragments thereof. The polymerase may be an alphavirus replicase, which comprises, for example, the alpha virus virus nsP4 protein.

While the natural alphavirus genomes encode structural virion proteins in addition to the non-structural replicase polyprotein, it is preferred that an alpha-virus-based self-replicating RNA molecule of the invention does not encode all the alphavirus structural proteins. Therefore, self-replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of Rv-containing alphavirus virions. The inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot be perpetuated infectiously. The structural proteins of the alphavirus that are necessary for perpetuation in natural viruses are absent from the self-replicating RNAs of the invention and their place is occupied by the gene or genes encoding the desired gene product (RSV protein or fragment thereof) ), so that the subgenomic transcript encodes the desired gene product instead of the structural proteins of the alphavirus virion.

Thus, a self-replicating RNA molecule useful with the invention has one or more sequences encoding RSV proteins or fragments thereof. The sequences encoding the RSV proteins or fragments can be in any desired orientation, and can be operably linked to the same promoters or to separate promoters. If desired, the sequences encoding the proteins or fragments of RSV can be part of a single open reading frame. In some embodiments, the RNA may have one or more additional open reading sequences or frames that encode, for example other additional RSV proteins or fragments thereof. A self-replicating RNA molecule can have a 5 'sequence that is compatible with the encoded replicase.

In one aspect, the self-replicating RNA molecule is derived from, or is based on, an alphavirus, such as the replicon of an alphavirus as defined herein. In other aspects, the self-replicating RNA molecule is derived or based on a virus other than an alphavirus, preferably a positive strand RNA virus, and more preferably a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus . Suitable natural alphavirus sequences are well known and available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru Virus (ATCC VR -600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya Virus (ATCC VR-64, ATCC VR-1241), Eastern Equine Encephalitis Virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah Virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro Virus (ATCC VR-66; ATCC VR-1277), Middleburg (ATCC VR-370 ), Mucambo Virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna Virus (ATCC VR-372, ATCC VR-1245), Ross River Virus (ATCC VR-373, ATCC VR -1246), Semliki Forest Virus (ATCC VR-67, ATCC VR-1247), Sindbis Virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), One (ATCC VR-374), Equine Encephalitis Virus of Venezuela (ATCC VR-69, ATCC VR-923, ATCC VR- 1250, ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis virus (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), e Y-62-33 (ATCC VR-375).

The self-replicating RNA molecules of the invention may contain one or more modified nucleotides and therefore have improved stability and are resistant to degradation and clearance in vivo, and other advantages. Without wishing to be bound by any theory, it is believed that self-replicating RNA molecules that contain modified nucleotides prevent or reduce stimulation of endosomal and cytoplasmic immune receptors when self-replicating RNA is administered in a cell. This allows self-replication, amplification and protein expression to occur. This also reduces safety risks with respect to the self-replicating RNA that does not contain modified nucleotides, because the self-replicating RNA that contains modified nucleotides reduces the activation of the innate immune system and subsequent unwanted consequences (e.g., inflammation at the injection site, injection site irritation, pain, and the like). It is also believed that the RNA molecules produced as a result of self-replication are recognized as foreign nucleic acids by cytoplasmic immune receptors. Therefore, self-replicating RNA molecules containing modified nucleotides provide effective amplification of RNA in a host cell and expression of CMV gB proteins, as well as adjuvant effects.

As used herein, "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (eg, substitutions) at or on the nitrogenous base of the nucleoside (eg, cytosine (C), thymine ( T) or uracil (U)), adenine (A) or guanine (G)). If desired, a self-replicating RNA molecule may contain chemical modifications in or on the sugar nucleoside moiety (eg, ribose, deoxyribose, modified ribose, modified deoxyribose, a six-member sugar analog, or a chain sugar analog open), or phosphate.

The self-replicating RNA molecules may contain at least one modified nucleotide, which is preferably not part of the 5 'cap. Accordingly, the self-replicating RNA molecule may contain a modified nucleotide in a single position, may contain a specific modified nucleotide (eg, pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine) in two or more positions, or it may contain two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides (for example, each in one or more positions). Preferably, the self-replicating RNA molecules comprise modified nucleotides that contain a modification on or in the nitrogen base, but do not contain modified sugar or phosphate moieties.

In some examples, between 0.001% and 99% or 100% of the nucleotides in a self-replicating RNA molecule are modified nucleotides. For example, 0.001% -25%, 0.01% -25%, 0.1% -25%, or 1% -25% of the nucleotides in a self-replicating RNA molecule are modified nucleotides.

In other examples, between 0.001% and 99% or 100% of a particular unmodified nucleotide in a self-replicating RNA molecule is replaced with a modified nucleotide. For example, about 1% of the nucleotides in the self-replicating RNA molecule that contain uridine may be modified, such as by replacing uridine with pseudouridine. In other examples, the desired amount (percentage) of two, three, or four specific nucleotides (nucleotides containing uridine, cytidine, guanosine, or adenine) in a self-replicating RNA molecule are modified nucleotides. For example, 0.001% -25%, 0.01% -25%, 0.1% -25, or 1% -25% of a particular nucleotide in a self-replicating RNA molecule are modified nucleotides. In other examples, 0.001% - 20%, 0.001% - 15%, 0.001% - 10%, 0.01% -20%, 0.01% -15%, 0.1% -25%, 0.01% -10%, 1% -20%, 1% -15%, 1% -10%, or approximately 5%, approximately 10%, approximately 15%, approximately 20% of a particular nucleotide in a molecule of Self-replicating RNAs are modified nucleotides.

It is preferred that less than 100% of the nucleotides in a self-replicating RNA molecule be modified nucleotides. It is also preferred that less than 100% of a particular nucleotide in a self-replicating RNA molecule be modified nucleotides. Therefore, preferred self-replicating RNA molecules comprise at least some unmodified nucleotides.

There are more than 96 naturally occurring nucleoside modifications found in mammalian RNA. See, for example, Limbach et al., Nucleic Acids Research, 22 (12): 2183-2196 (1994). The preparation of modified nucleotides and nucleotides and nucleosides is well known in the art, for example, from United States patents Nos. 4373071,4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642, and are commercially available many modified nucleosides and modified nucleotides.

Modified nucleobases that can be incorporated into modified nucleosides and modified nucleotides and are present in self-replicating RNA molecules include m5C (5-methylcitidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine) , Um (2'-0-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-0-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6- (cis-hydroxyisopentenyl) adenosine); ms2io6A (2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar (p) (2'-O-ribosiladenosine (phosphate)); I (inosine); m11 (1-methylinosine); m'lm (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcitidine); m5Cm (5,2-0-dimethyl-citidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lisidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2, N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2, N2,2'-O-trimethylguanosine); Gr (p) (2'-O-ribosylguanosine (phosphate)); yW (wibutosine); o2yW (peroxywibutosine); OHyW (hydroxywibutosine); OHyW * (unmodified hydroxywibutosine); imG (wiosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyiqueuosin); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-desazaguanosine); preQi (7-aminomethyl-7-desazaguanosine); G (arcaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3- (3-amino-3-carboxypropyl) uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyxyuridine); cmo5U (5-oxyacetic uridine acid); mcmo5U (5-oxyacetic acid uridine methyl ester); chm5U (5- (carboxyhydroxymethyl) uridine)); mchm5U (5- (carboxyhydroxymethyl) uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6, N6-dimethyladenosine); Tm (2'-O-methlinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6, TO-dimethyladenosine); rn62Am (N6, N6, O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2, N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); m1Gm (1,2'-O-dimethylguanosine);m'Am (1,2-0-dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14 (4-desmethyl guanosine); imG2 (isoguanosine); or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7 substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-alkyluracil (C ¹ -C ⁶ ), 5-methyluracil, 5-alkenyluracil (C ² -C ⁶ ), 5-alkynyluracil (C ² -C ⁶ ), 5- (hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine , 5-alkylcytosine (C ¹ -C ⁶ ), 5-methylcytoxin, 5-alkenylcytosine (C ² -C ⁶ ), 5-alkynylcytosine (C ² -C ⁶ ), 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-desazaguanine 7-substituted, 7-desaza-7-alkynylguanine (C2-C6), 7-desazaguanine-8-substituted, 8-hydroxyguanine, 6-thioguanine, 8- oxoguanin, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7 dezapurine, 7-diszapurine 7 substituted, 7-desazapurine-8-substituted, and hydrogen (abbasic rest). m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. Any one or any combination of these modified nucleobases may be included in the self-replicating RNA of the invention. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers.

If desired, the self-replicating RNA molecule may contain phosphoramidate, phosphorothioate, and / or methyl phosphonate loops.

Self-replicating RNA molecules comprising at least one modified nucleotide can be prepared using any suitable procedure. Some methods suitable for producing RNA molecules containing modified nucleotides are known in the art. For example, a self-replicating RNA molecule containing modified nucleotides can be prepared by transcribing (eg, by transcription ( in vitro) a DNA encoding the self-replicating RNA molecule using a suitable DNA-dependent RNA polymerase, such as an RNA polymerase from the phage T7, phage RNA polymerase SP6, phage RNA polymerase T3, and the like, or mutants of these polymerases that allow the effective incorporation of modified nucleotides into RNA molecules.The transcription reaction will contain modified nucleotides and nucleotides, and other components which support the activity of the selected polymerase, such as a suitable buffer, and the appropriate salts, the incorporation of nucleotide analogs into a self-replicating RNA can be engineered, for example, to alter the stability of said RNA molecules, for increase resistance against RNAse, to establish the replica tion after introduction into suitable host cells ("infectivity" of the RNA), and / or to induce or reduce innate and adaptive immune responses.

Suitable synthetic procedures may be used alone, or in combination with one or more different procedures (eg, recombinant DNA or RNA technology), to produce a self-replicating RNA molecule that contains one or more modified nucleotides. Suitable procedures for de novo synthesis are well known in the art and can be adapted for specific applications. Illustrative procedures include, for example, chemical synthesis using suitable protecting groups such as CEM (Masuda et al. (2007) Nucleic Acids Symposium Series 51: 3-4), the p-cyanoethyl phosphoramidite (Beaucage SL and col. (1981) Tetrahedron Lett 22: 1859); the H-phosphonate nucleoside procedure (Garegg P et al. (1986) Tetrahedron Lett 27: 4051-4; Froehler BC et al. (1986) Nucl Acid Res 14: 5399-407; Garegg P et al. (1986) Tetrahedron Lett 27: 4055-8; Gaffney BL et al. (1988) Tetrahedron Lett 29: 2619-22). These chemicals can be carried out or adapted for use with automated nucleic acid synthesizers that are commercially available. Further suitable synthetic procedures are disclosed in Uhlmann et al. (1990) Chem Rev 90: 544-84, and Goodchild J (1990) Bioconjugate Chem 1: 165. Nucleic acid synthesis can also be carried out using suitable recombinant methods that are well known and conventional in the art, including cloning, processing, and / or expression of polynucleotides and gene products encoded by said polynucleotides. Transposition of DNA by random fragmentation and reassembly of gene fragments and synthetic polynucleotides by PCR are examples of known techniques that can be used to design and design genetically engineered polynucleotide sequences. Site-directed mutagenesis can be used to alter nucleic acids and encoded proteins, for example, to insert new restriction sites, alter glycosylation models, change codon preferences, produce splicing variants, introduce mutations and Similar. Suitable methods for transcription, translation and expression of nucleic acid sequences are known, and are conventional in the art. (see generally, Current Protocols in Molecular Biology, Vol.

2, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986; Bitter, et al., In Methods in Enzymology 153: 516-544 (1987); The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II, 1982; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.)

The presence and / or quantity of one or more modified nucleotides in a self-replicating RNA molecule can be determined using any suitable procedure. For example, a self-replicating RNA can be digested to monophosphates (for example, using P1 nuclease) and autophosphorylated (for example, using a suitable phosphatase such as CIAP) and the resulting nucleosides can be analyzed by reverse phase HPLC (using, for example, a column YMC Pack ODS-AQ (5 micrometers, 4.6 X 250 M) and eluted using a gradient, 30% B (0-5 min) at 100% B (5 - 13 min) and 100% B (13 -40) min, flow rate (0.7 ml / min), UV detection (wavelength: 260 nm), column temperature (30 ° C) Buffer A (20 mM acetic acid - ammonium acetate pH 3, 5), buffer B (20 M acetic acid - ammonium acetate pH 3.5 / methanol [90/10])).

The self-replicating RNA can be associated with an administration system. Self-replicating RNA can be administered with or without an adjuvant.

RNA management systems

The self-replicating RNAs described herein are suitable for administration in a variety of modalities, such as the administration of pure RNA or in combination with lipids, polymers or other compounds that facilitate entry into cells. Self-replicating RNA molecules can be introduced into cells or target subjects using any suitable technique, for example, by direct injection, microinjection, electroporation, lipofection, biolistics, and the like. The self-replicating RNA molecule can also be introduced into cells by means of receptor-mediated endocytosis. See, for example, U.S. Patent No. 6,090,619; Wu and Wu, J.

Biol. Chem., 263: 14621 (1988); and Curiel et al., Proc. Natl Acad. Sci. USA, 88: 8850 (1991). For example, U.S. Patent No. 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by associating the nucleic acid with a polycation moiety (for example, poly-L-lysine having 3-100 lysine residues (SEQ ID NO: 19)), which itself is coupled to an integrin receptor binding moiety (for example, a cyclic peptide having the Arg-Gly-Asp sequence (SEQ ID NO: 16).

Self-replicating RNA molecules can be administered in cells by amphiphiles. See, for example, U.S. Patent No. 6,071,890. Normally, a nucleic acid molecule can complex with the cationic amphiphile. Mammalian cells that have come into contact with the complex can easily capture this.

The self-replicating RNA can be administered as pure RNA (for example, merely as an aqueous RNA solution) but, to enhance the entry into the cells and also the subsequent intercellular effects, the self-replicating RNA is preferably administered in combination with an administration system, such as a particulate or emulsion administration system. A large number of administration systems are well known to those skilled in the art. Such administration systems include, for example, liposome-based administration (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6 (7): 682-691; Rose US Pat. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413 7414), as well as the use of viral vectors for example, adenovirals (see, for example, Berns et al. (1995) Ann. NY Acad. Sci. 772: 95-104; Ali et al. (1994) Gene Ther. 1: 367-384; and Haddada et al. (1995) Curr. Top Microbiol Immunol

199 (Pt 3): 297-306 for a review), papillomavirus, retroviral (see, for example, Buchscher et al. (1992) J. Virol. 66 (5) 2731-2739; Johann et al. (1992) J. Virol. 66 (5): 1635-1640 (1992); Sommerfelt et al. (1990) Virol. 176: 58-59; Wilson et al. (1989) J. Virol. 63: 2374-2378; Miller et al. ., J. Virol. 65: 2220-2224 (1991); Wong-Staal et al., PCT / US94 / 05700, and Rosenburg and Fauci (1993) in Fundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd. , New York and previous references, and Yu et al., Gene Therapy (1994) above.), And adeno-associated viral vectors (See, West et al. (1987) Virology 160: 38-47; Carter et al. ( 1989) U.S. Patent No. 4,797,368; Carter et al. Document w O 93/24641 (1993); Kotin (1994) Human Gene Therapy 5: 793-801; Muzyczka (1994) J. Clin. Invst. 94: 1351 and Samulski (later) for a review of adenoviral vectors; see also, Lebkowski, U.S. Patent No. 5,173,414; Tratsch in et al. (1985) Mol. Cell Biol. 5 (11): 3251-3260; Tratschin, et al. (1984) Mol. Cell Biol., 4: 2072-2081; Hermonat and Muzyczka (1984) Proc. Natl Acad. Sci. USA, 81: 6466-6470; McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol., 63: 03822-3828), and the like.

Three particularly useful delivery systems are (i) liposomes, (ii) microparticles of non-toxic and biodegradable polymers, and (iii) cationic submicronic oil in water emulsions.

Liposomes

Various amphiphilic lipids can form bilayers in an aqueous environment to encapsulate an aqueous core that contains an RNA such as a liposome. These lipids may have an anionic, cationic or hybrid ion head group. The formation of liposomes from anionic phospholipids dates back to the 1960s, and cationic liposome-forming lipids have been studied since the 1990s. Some phospholipids are anionic while others are of hybrid ions. Suitable classes of phospholipids include, but are not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols. Useful cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), 1,2-distearyloxy-N, N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N, Ndimethyl-3- aminopropane (d Od MA), 1,2-dilinoleyloxy-N, N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethyl-3-aminopropane (DLenDMA). Hybrid ion lipids include, but are not limited to, acyl hybrid ion lipids and ether hybrid ion lipids. Examples of useful hybrid ion lipids are DPPC, DOPC and dodecylphosphocholine. Lipids may be saturated or unsaturated.

Liposomes can be formed from a single lipid or from a mixture of lipids. A mixture may comprise (i) a mixture of anionic lipids (ii) a mixture of cationic lipids (iii) a mixture of hybrid ion lipids (iv) a mixture of anionic lipids and cationic lipids (v) a mixture of anionic lipids and Hybrid ion lipids (vi) a mixture of hybrid ion lipids and cationic lipids or (vii) a mixture of anionic lipids, cationic lipids and hybrid ion lipids. Similarly, a mixture may comprise saturated and unsaturated lipids. For example, a mixture may comprise DSPC (hybrid ion, saturated), DlinDMA (cationic, unsaturated), and / or DMPG (anionic, saturated). When a lipid mixture is used, not all component lipids in the mixture need to be amphiphilic, for example, one or more amphiphilic lipids can be mixed with cholesterol.

The hydrophilic part of a lipid may be PEGylated (ie, modified by covalent bonding of a polyethylene glycol). This modification can increase stability and prevent nonspecific adsorption of liposomes. For example, lipids can be conjugated with PEG using techniques such as those disclosed in Heyes et al. (2005) J Controlled Release 107: 276-87.

A mixture of DSPC, DlinDMA, PEG-DMPG and cholesterol can be used to form liposomes. A separate aspect of the invention is a liposome comprising DSPC, DlinDMA, PEG-DMG and cholesterol. This liposome preferably encapsulates RNA, such as a self-replicating RNA, for example, that encodes an immunogen.

Liposomes are usually divided into three groups: multilamellar vesicles (MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles (LUV). MLVs have multiple bilayers in each vesicle, forming some separate aqueous compartments. SUVs and LUVs have a single bilayer that encapsulates an aqueous core; The s Uv normally have a diameter <50 nm, and the LUV have a diameter> 50 nm. Liposomes useful with the invention without ideally LUVs with a diameter in the range of 50-220 nm. For a composition comprising a population of LUV with different diameters: (i) at least 80% in number must have diameters in the range of 20-220 nm, (ii) the average diameter (Zav, by intensity) of the population It is ideally in the range of 40-200 nm, and / or (iii) the diameters should have a polydispersity index <0.2.

Techniques for preparing suitable liposomes are well known in the art, for example, see Liposomes: Methods and Protocols, Volume 1: Pharmaceutical Nanocarriers: Methods and Protocols. (ed. Weissig). Humana Press, 2009. ISBN 160327359X; Liposome Technology, volumes I, II and III. (ed. Gregoriadis). Informa Healthcare, 2006; and Functional Polymer Colloids and Microparticles volume 4 (Microspheres, microcapsules & liposomes). (eds. Arshady and Guyot). Citus Books, 2002. A useful procedure involves mixing (i) an ethanolic solution of the lipids (ii) in an aqueous solution of the nucleic acid and (iii) buffer, followed by mixing, equilibrium, dilution and purification (Heyes et al. ( 2005) J Controlled Release 107: 276-87.).

RNA is preferably encapsulated in liposomes, and therefore, the liposome forms an outer layer around a nucleus that contains an aqueous RNA solution. It has been found that this encapsulation protects the RNA from the RNAse digestion. The liposomes may include some external RNA (for example, on the surface of the liposomes), but preferably, at least half of the RNA (and ideally virtually everything) is encapsulated.

Polymeric microparticles

Various polymers can form microparticles to encapsulate or adsorb RNA. The use of a substantially non-toxic polymer means that the receptor can safely receive the particles, and the use of a biodegradable polymer means that the particles can be metabolized after administration to avoid long-term persistence. Useful polymers are also sterilizable, to aid in pharmaceutical quality preparations.

Suitable non-toxic and biodegradable polymers include, but are not limited to, poly (a-hydroxy acids), polyhydroxybutyric acids, polylactones (including polycaprolactones), polydioxanones, polyhydrolactones, polyorthoesters, polyanhydrides, polycyanoacrylates, polycarbonates derived from tyrosine, tyrosine derivatides, tyrosine derivatives and combinations thereof.

In some embodiments, the microparticles are formed from poly (a-hydroxy acids), such as poly (lactides) ("PLA"), lactide copolymers and glycolide such as poly (D, L-lactide-co-glycolide) ( "PLG"), and copolymers of D, L-lactide and caprolactone. Useful PLG polymers include those that have a lactide / glycolide molar ratio ranging, for example, from 20:80 to 80:20, for example, 25:75, 40:60, 45:55, 55:45, 60:40, 75:25. Useful PLG polymers include those having a molecular weight between, for example, 5,000-200,000 Da, for example, between 10,000 100,000, 20,000-70,000, 40,000-50,000 Da.

The microparticles ideally have a diameter in the range of 0.02 pm to 8 pm. For a composition comprising a population of microparticles with different diameters, at least 80% in number must have diameters in the range of 0.03-7 pm.

Techniques for preparing suitable microparticles are well known in the art, for example, see Functional Polymer Colloids and Microparticles volume 4 (Microspheres, microcapsules & liposomes). (eds. Arshady and Guyot). Citus Books, 2002; Polymers in Drug Delivery. (eds. Uchegbu & Schatzlein). CRC Press, 2006. (in particular chapter 7) and Microparticulate Systems for the Delivery of Proteins and Vaccines. (eds. Cohen & Bernstein). CRC Press, 1996. To facilitate the adsorption of RNA, a microparticle may include a cationic surfactant and / or a lipid, for example, as disclosed in O'Hagan et al. (2001) J Virology75: 9037-9043; and Singh et al. (2003) Pharmaceutical Research 20: 247-251. An alternative way of preparing polymeric microparticles is by molding and curing, for example, as disclosed in WO2009 / 132206.

The microparticles of the invention can have a zeta potential of between 40-100 mV. RNA can be absorbed in the microparticles, and absorption is facilitated by the inclusion of cationic materials, (eg, cationic lipids) in the microparticle.

Cationic emulsions of oil in water

Oil-in-water emulsions are known to help with flu vaccines, for example, MF59 ™ adjuvant in FLUAD ™ product, and AS03 adjuvant in PREPANDRIX ™ product. The administration of the RNA can be carried out with the use of an oil-in-water emulsion, with the proviso that the emulsion includes one or more cationic molecules. For example, a cationic lipid may be included in the emulsion to provide a positively charged surface of a droplet to which the negatively charged RNA can bind.

The emulsion comprises one or more oils. Suitable oils include those from, for example, an animal (such as a fish) or a plant source. The oil is ideally biodegradable (metabolizable) and biocompatible Sources of vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, illustrates nut oils. Jojoba oil can be used, for example, obtained from jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the group of grain oils, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, tritical and the like can also be used. The fatty acid esters of 6 10 carbon atoms of glycerol and 1,2-propanedyl, although not naturally occurring in seed oils, can be prepared by hydrolysis, separation and esterification of suitable materials starting from nut oils and seeds. Fats and oils of mammalian milk are metabolized and can therefore be used. The separation, purification, saponification and other means necessary to obtain pure oils from animal sources are well known in the art.

Most fish contains metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti illustrates some of the fish oils that can be used herein. Numerous branched chain oils are biochemically synthesized in isoprene units of 5 carbon atoms and are generally referred to as terpenoids. Squalane, the saturated analog of squalene, can also be used. Fish oils, including squalene and squalane, are readily available from commercial sources or can be obtained by methods known in the art.

Other useful oils are tocopherols, particularly in combination with squalene. When the oil phase of an emulsion includes a tocopherol, any of the tocopherols a, p, and, 5, £ or, ^ may be used but the tocopherols a are preferred. D-a-tocopherol and DL-a-tocopherol can be used. An a-tocopherol a is DL-a-tocopherol. A combination of oil comprising squalene and a tocopherol (for example, DL-a-tocopherol) can be used.

Preferred emulsions comprise squalene, a shark liver oil that is a branched unsaturated terpenoid (C ³⁰ H ⁵⁰ ; [(CH ³ ) ² C [= CHCH ² CH ² C (CHa)] ² = CHCH ² -] ² ; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane; CAS RN 7683-64-9).

The oil in the emulsion may comprise a combination of oils, for example, squalene and at least one additional oil.

The aqueous component of the emulsion may be tap water (for example, water for injection) or may include additional components, for example, solutes. For example, it may include salts to form a buffer, for example, citrate or phosphate salts, such as sodium salts. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. A buffered aqueous phase is preferred, and the buffers will normally be included in the range of 5-20 mM.

The emulsion also includes a cationic lipid. Preferably, this lipid is a surfactant so that it can facilitate the formation and stabilization of the emulsion. Useful cationic lipids generally contain a nitrogen atom that is positively charged under physiological conditions, for example, as a tertiary or quaternary amine. This nitrogen may be in the hydrophilic head group of an amphiphilic surfactant. Useful cationic lipids include, but are not limited to: 1,2-dioleoyloxy-3- (trimethylammonium) propane (DOTAP), 3 '- [N- (N', N'-Dimethylaminoethane) -carbamoyl] cholesterol (DC Cholesterol ), dimethyldioctadecyl ammonium (DDA, for example, bromide), 1,2-Dimiristoyl-3-trimethyl-ammonium propane (DMTAP), dipalmitoyl (C16: 0) trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP). Other useful cationic lipids are: benzalkonium chloride (BAK), benzethonium chloride, cetramide (containing tetradecyltrimethylammonium bromide and possibly small amounts of dedecyltrimethylammonium bromide and hexadecyltrimethyl ammonium bromide), cetylpyridinium chloride (CPC), cetyl chloride trimethylammonium (CTCA), N, N ', N'-polyoxyethylene (10) -N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, mixed with alkyltrimethylammonium bromide, benzyl dimethyldodecylammonium bromide, dimethyl ammonium bromide benzyltrimethylammonium methoxide, cetyl dimethyl ethyl ammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decametonium chloride, methyl chloride mixed with trialkylammonium, methyl trioctyl ammonium chloride), N, N-dimethyl-N- [2 ( 2-methyl-4- (1,1,3,3-tetramethylbutyl) -phenoxy] -ethoxy) ethyl] -benzenemethanamine (DEBDA), dialkyl dimethylammonium salts, [1- (2,3 -dioleyloxy) -propyl] -N, N, N, trimethylammonium, 1,2-diacyl-3- (trimethylammonium) propane (acyl group = dimiristoyl, dipalmitoyl, distearoyl, dioleoyl), l, 2-diacyl-3 (dimethylammonium) propane (acyl group = dimiristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-3- (4'-trimethyl-ammonium) butanoyl-sn-glycerol, 1,2-dioleoyl ester 3-succinyl-sn-glycerol choline, cholesteryl (4'-trimethylammonium) butanoate), N-alkylpyridinium salts (eg, cetylpyridinium bromide and cetylpyridinium chloride), N-alqulpiperidinium salts, dicathionic ballyl electrolytes (C12Me6; C12BU6), dialkylglycylphosphorylcholine, lysolecithin, Dioleoylphosphatidylethanolamine, cholesterol ester choline hemisuccinate, lipopolyamines, including but not limited to dioctadecylmidoglycyl spermine (DOGS), dipalmitoyl phosphatidylethanolamido-L-dperpyl (DPL) LPL (dlp) L-dlp (LPL) LPL (dlp) LP-lysperine (DP) , poly (L (or D) -lisin conjugated to N-glutarylphosphatidylethanolamine, didodecyl glutamate ester with a pendant amino group (CAGluPhCnN), ditetradecyl glutamate ester with pendant amino group (Cl4GIuCnN +), cholesterol cationic derivatives, including, although not limited to the salt of cholesteryl-3 p-oxisuccinamidoethylenetrimethylammonium, cholesteryl-3 poxisuccinamidoethylene dimethylamine, salt of cholesteryl-3 p-carboxyamidoethylenetrimethylammonium, and cholesterol-3 pcarboxyamidoethylenethioethyl ammonium 8,88th. Y US 2008/0057080. The cationic lipid is preferably biodegradable (metabolizable) and biocompatible.

In addition to oil and cationic lipid, an emulsion may include a nonionic surfactant and / or a hybrid ion surfactant. Such surfactants include, but are not limited to: polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), marketed under the trade name DOWFAX ™, such as linear EO / PO block copolymers; octoxinoles, which may vary in the number of the ethoxy repeating groups (oxy-1,2-ethanediyl), with octoxynol-9 (Triton X-100, or toctylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); polyoxyethylene lauryl ether 9; and sorbitan esters (commonly known as Spans), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants to include in the emulsion are polysorbate 80 (Tween 80; polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.

Mixtures of these surfactants may be included in the emulsion, for example, mixtures of Tween 80 / Span 85, or mixtures of Tween 80 / Triton-X100. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol. Useful mixtures may comprise a surfactant with an HLB value in the range of 10-20 (for example, polysorbate 80, with an HLB of 15.0) and a surfactant with an HLB value in the range of 1-10 (for example , sorbitan trioleate, with an HLB of 1.8).

Preferred amounts of oil (% by volume) in the final emulsion are between 2-20%, for example, 5-15%, 6-14%, 7-13%, 8-12%. A squalene content of about 4-6% or about 9-11% is particularly useful.

Preferred amounts of surfactants (% by weight) in the final emulsion are between 0.001% and 8%. For example: polyoxyethylene sorbitan esters (such as polysorbate 80) 0.2 to 4%, in particular between 0.4-0.6%, between 0.45 0.55%, about 0.5% or between 1 , 5-2%, between 1.8-2.2%, between 1.9-2.1%, approximately 2%, or 0.85 0.95%, or approximately 1%; sorbitan esters (such as sorbitan trioleate) 0.02 to 2%, in particular about 0.5% or about 1%; octyl- or nonylphenoxy polyoxyethanes (such as Triton X-100) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 8%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

The absolute amounts of oil and surfactant, and their ratio, can be varied within wide limits although still forming an emulsion. An expert person can easily vary the relative proportions of the components to obtain a desired emulsion, but a weight ratio of between 4: 1 and 5: 1 is typical for the oil and the surfactant (excess oil).

An important parameter to ensure the immunostimulatory activity of an emulsion, particularly in large animals, is the size of the oil droplet (diameter). The most effective emulsions have an oil droplet size in the submicron range. Suitably, the droplet sizes will be in the range of 50-750 nm. More usefully, the average droplet size is less than 250 nm, for example, less than 200 nm, less than 150 nm. The average droplet size is useful in the range of 80-180 nm. Ideally, at least 80% (in number) of the droplets of the emulsion oil are less than 250 nm in diameter, and preferably at least 90%. Apparatus for determining the average droplet size in an emulsion, and the size distribution, are commercially available. these normally use the techniques of dynamic light scattering and / or the optical detection of individual particles, for example, by the Accusizer ™ and Nicomp ™ series of instruments available from Particle Sizing Systems (Santa Barbara, USA), or Zetasizer instruments ™ from Malvern Instruments (United Kingdom) or the Horiba particle size distribution Analyzer instruments (Kyoto, Japan).

Ideally, the distribution of droplet sizes (by number) has only a maximum, that is, there is a single population of droplets distributed around an average (mode), rather than having two maxima. Preferred emulsions have a polydispersity of <0.4, for example, 0.3, 0.2, or less.

Suitable emulsions with submicron droplets and a narrow size distribution can be obtained through the use of microfluidization. This technique reduces the average oil droplet size by propelling the currents of the inlet elements through geometrically fixed channels at high pressure and high speed. These currents come into contact with the walls of the channels, the walls of the chamber and each other. The results of shear, impact and cavitation forces produce a reduction in droplet size. Repeated microfluidization steps can be carried out until an emulsion with a desired average droplet size and distribution is achieved.

As an alternative to microfluidization, thermal procedures can be used to produce a phase inversion. These procedures can also provide a submicron emulsion with a distribution of narrow particle size.

Preferred emulsions can be filtered sterilized, that is, their droplets can pass through a 220 nm filter. As well as providing sterilization, this procedure also removes any large droplets in the emulsion.

In certain embodiments, the cationic lipid in the emulsion is DOTAP. The oil-in-cationic water emulsion may comprise between about 0.5 mg / ml about 25 mg / ml of DOTAP. For example, the oil-in-cationic emulsion may comprise DOTAP at between about 0.5 mg / ml to about 25 mg / ml, from about 0.6 mg / ml to about 25 mg / ml, from about 0.7 mg / ml to approximately 25 mg / ml, approximately 0.8 mg / ml to approximately 25 mg / ml, approximately 0.9 mg / ml to approximately 25 mg / ml, approximately 1.0 mg / ml to approximately 25 mg / ml, from approximately 1.1 mg / ml to approximately 25 mg / ml, from approximately 1.2 mg / ml to approximately 25 mg / ml, from approximately 1.3 mg / ml to approximately 25 mg / ml, of approximately 1.4 mg / ml to approximately 25 mg / ml, approximately 1.5 mg / ml to approximately 25 mg / ml, approximately 1.6 mg / ml to approximately 25 mg / ml, approximately 1.7 mg / ml at about 25 mg / ml, from about 0.5 mg / ml to about 24 mg / ml, from about 0.5 mg / ml to about 22 mg / ml, from about 0.5 mg / ml to about 20 mg / ml, of ap approximately 0.5 mg / ml to approximately 18 mg / ml, approximately 0.5 mg / ml to approximately 15 mg / ml, approximately 0.5 mg / ml to approximately 12 mg / ml, approximately 0.5 mg / ml to approximately 10 mg / ml, from approximately 0.5 mg / ml to approximately 5 mg / ml, from approximately 0.5 mg / ml to approximately 2 mg / ml, from approximately 0.5 mg / ml to approximately 1 , 9 mg / ml, from approximately 0.5 mg / ml to approximately 1.8 mg / ml, from approximately 0.5 mg / ml to approximately 1.7 mg / ml, from approximately 0.5 mg / ml to approximately 1.6 mg / ml, from approximately 0.6 mg / ml to approximately 1.6 mg / ml, from approximately 0.7 mg / ml to approximately 1.6 mg / ml, from approximately 0.8 mg / ml to approximately 1.6 mg / ml, approximately 0.5 mg / ml, approximately 0.6 mg / ml, approximately 0.7 mg / ml, approximately 0.8 mg / ml, approximately 0.9 mg / ml, approximately 1 , 0 mg / ml, approximately 1.1 mg / ml, approximately 1.2 mg / ml, approximately 1.3 mg / ml, approximately 1.4 mg / ml, approximately 1.5 mg / ml, approximately 1.6 mg / ml, approximately 12 mg / ml, approximately 18 mg / ml, approximately 20 mg / ml, approximately 21.8 mg / ml, approximately 24 mg / ml, etc. In an illustrative embodiment, the cationic water oil emulsion comprises from about 0.8 mg / ml to about 1.6 mg / ml of DOTAP, such as 0.8 mg / ml, 1.2 mg / ml, 1, 4 mg / ml or 1.6 mg / ml.

In certain embodiments, the cationic lipid is DC cholesterol. The oil emulsion in cationic water may comprise DC Cholesterol at between about 0.1 mg / ml to about 5 mg / ml of DC Cholesterol. For example, the oil emulsion in cationic water may comprise DC cholesterol between about 0.1 mg / ml to about 5 mg / ml, from about 0.2 mg / ml to about 5 mg / ml, from about 0.3 mg / ml to approximately 5 mg / ml, from approximately 0.4 mg / ml to approximately 5 mg / ml, from approximately 0.5 mg / ml to approximately 5 mg / ml, from approximately 0.62 mg / ml to approximately 5 mg / ml, approximately 1 mg / ml to approximately 5 mg / ml, approximately 1.5 mg / ml to approximately 5 mg / ml, approximately 2 mg / ml to approximately 5 mg / ml, approximately 2.46 mg / ml to approximately 5 mg / ml, from approximately 3 mg / ml to approximately 5 mg / ml, from approximately 3.5 mg / ml to approximately 5 mg / ml, from approximately 4 mg / ml to approximately 5 mg / ml , from about 4.5 mg / ml to about 5 mg / ml, from about 0.1 mg / ml to about 4.92 mg / ml, from about 0.1 mg / ml to about 4.5 mg / ml, of approximate 0.1 mg / ml to approximately 4 mg / ml, from approximately 0.1 mg / ml to approximately 3.5 mg / ml, from approximately 0.1 mg / ml to approximately 3 mg / ml, from approximately 0, 1 mg / ml to approximately 2.46 mg / ml, from approximately 0.1 mg / ml to approximately 2 mg / ml, from approximately 0.1 mg / ml to approximately 1.5 mg / ml, from approximately 0.1 mg / ml ml to approximately 1 mg / ml, approximately 0.1 mg / ml to approximately 0.62 mg / ml, approximately 0.15 mg / ml, approximately 0.3 mg / ml, approximately 0.6 mg / ml, approximately 0.62 mg / ml, approximately 0.9 mg / ml, approximately 1.2 mg / ml, approximately 2.46 mg / ml, approximately 4.92 mg / ml, etc. In an illustrative embodiment, the cationic water oil emulsion comprises between about 0.62 mg / ml to about 4.92 mg / ml of DC Cholesterol, such as 2.46 mg / ml.

In certain embodiments, the cationic lipid is DDA. The cationic water oil emulsion may comprise between about 0.1 mg / ml to about 5 mg / ml DDA. For example, the oil-in-cationic emulsion may comprise DDA at between about 0.1 mg / ml to about 5 mg / ml, from about 0.1 mg / ml to about 4.5 mg / ml, from about 0, 1 mg / ml to about 4 mg / ml, from about 0.1 mg / ml to about 3.5 mg / ml, from about 0.1 mg / ml to about 3 mg / ml, from about 0.1 mg / ml to approximately 2.5 mg / ml, approximately 0.1 mg / ml to approximately 2 mg / ml, approximately 0.1 mg / ml to approximately 1.5 mg / ml, approximately 0.1 mg / ml at about 1.45 mg / ml, from about 0.2 mg / ml to about 5 mg / ml, from about 0.3 mg / ml to about 5 mg / ml, from about 0.4 mg / ml to about 5 mg / ml, from approximately 0.5 mg / ml to approximately 5 mg / ml, from approximately 0.6 mg / ml to approximately 5 mg / ml, from approximately 0.73 mg / ml to approximately 5 mg / ml, of approximately 0.8 mg / ml to approximately 5 mg / ml, approximately about 0.9 mg / ml at approximately 5 mg / ml, from about 1.0 mg / ml to about 5 mg / ml, from about 1.2 mg / ml to about 5 mg / ml, from about 1.45 mg / ml to about 5 mg / ml, from about 2 mg / ml at approximately 5 mg / ml, from approximately 2.5 mg / ml to approximately 5 mg / ml, from approximately 3 mg / ml to approximately 5 mg / ml, from approximately 3.5 mg / ml to approximately 5 mg / ml, approximately 4 mg / ml to approximately 5 mg / ml, approximately 4.5 mg / ml to approximately 5 mg / ml, approximately 1.2 mg / ml, approximately 1.45 mg / ml, etc. Alternatively, the oil-in-cationic emulsion can comprise DDA at about 20 mg / ml, about 21 mg / ml, about 21.5 mg / ml, about 21.6 mg / ml, about 25 mg / ml. In an illustrative embodiment, the cationic water oil emulsion comprises between about 0.73 mg / ml to about 1.45 mg / ml of DDA, such as 1.45 mg / ml.

Catheters or similar devices can be used to administer the self-replicating RNA molecules of the invention, as pure RNA or in combination with an administration system, in a target organ or tissue. Suitable catheters are disclosed in, for example, US Pat. No. 4,186,745; 5,397,307; 5,547,472; 5,674,192; and 6,129,705.

The present invention includes the use of suitable delivery systems, such as liposomes, polymeric microparticles or microparticles of submicron emulsions with encapsulated or adsorbed self-replicating RNA, to administer a self-replicating RNA molecule that encodes two or more v Rs proteins, for example, to stimulate a single immune response, or in combination with another macromolecule. The invention includes liposomes, microparticles and submicron emulsions with adsorbed and / or encapsulated self-replicating RNA molecules, and combinations thereof.

The self-replicating RNA molecules associated with liposomes and microparticles of submicron emulsions can be effectively administered to a host cell, and can induce an immune response in the protein encoded by the self-replicating RNA.

Polycistronic self-replicating RNA molecules encoding the VRS protein, and the VRPs produced using polycistronic alphavirus replicons, can be used to form VRS protein complexes in a cell. In some embodiments, combinations of VRP or VPR containing sequences encoding two or more proteins or fragments of RSV are administered to a cell. In some embodiments, combinations of the self-replicating RNA molecules or self-replicating RNA molecules encoding two or more RSV proteins or fragments are administered to a cell.

The inducing composition of the immune response directed against RSV

The compositions inducing an immune response directed against RSV may comprise one or more immunoregulatory agents. Preferably, one or more of the immunoregulatory agents include one or more adjuvants, for example two, three, four or more adjuvants. The adjuvants may include an adjuvant TH1 and / or an adjuvant t H2, described in more detail below.

The compositions of the invention may include one or more pharmaceutically acceptable carriers and / or excipients, including adjuvants. An exhaustive description of these components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. The compositions will generally be in aqueous form.

The composition may include preservatives, such as thimerosal or 2-phenoxyethanol. It is preferred, however, that the vaccine should be substantially free (i.e., less than 5 pg / ml) of mercuric material, for example, free of thiomersal. Immunogenic compositions that do not contain mercury are more preferred. Preservative-free immunogenic compositions are particularly preferred.

To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl), which may be present at between 1 and 20 mg / ml, is preferred. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, dehydrated disodium phosphate, magnesium chloride, calcium chloride, and the like.

The compositions will generally have an osmolality of between 200 mOsm / kg and 400 mOsm / kg, preferably, between 240-360 mOsm / kg and more preferably, will be in the range of 290-310 mOsm / kg.

The compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (in particular, with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will normally be included in the 5-20 mM range. The pH of the composition will generally be between 5.0 and 8.1 and more typically between 6.0 and 8.0, for example, between 6.5 and 7.5, or between 7.0 and 7.8. A method of the invention can therefore include a step of adjusting the pH of the volumetric vaccine before packaging.

The composition is preferably sterile. The composition is preferably non-pyrogenic, for example, containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. The composition It is preferably gluten free. Vaccines for humans are normally administered in a dose volume of approximately 0.5 ml, although half doses (ie approximately 0.25 ml) can be administered to children.

Compositions of the invention, which contain VRS-F polypeptides, or nucleic acids encoding VRS-F polypeptides, may also include one or more adjuvants, for example two, three, four or more adjuvants, which may function to enhance responses. immune (humoral and / or cellular) stimulated in a patient receiving the composition. The adjuvants may include a TH1 adjuvant and / or a TH2 adjuvant. Adjuvants that can be used in the compositions of the invention include, but are not limited to:

• Compositions containing minerals. Compositions containing minerals suitable for use as an adjuvant in the invention include mineral salts, such as calcium salts and aluminum salts (or mixtures thereof). The invention includes mineral salts such as hydroxides (for example, oxyhydroxides), phosphates (for example, hydroxyphosphates, orthophosphates), sulfates, etc., or mixtures of different mineral compounds, with compounds that take any suitable form (for example, form gel, crystalline, amorphous, etc.), and adsorption being preferred. Calcium salts include calcium phosphate (for example, the "CAP" particles disclosed in US Patent No. 6355271). Aluminum salts include hydroxides, phosphates, sulfates, and the like. Mineral-containing compositions can also be formulated as a particle of a metal salt (WO00 / 23105). Adjuvants of aluminum salts are described in more detail below.

• Compositions of oily emulsions (see in more detail below). Compositions of oily emulsions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5% squalene, 0.5% Tween 80 and 0.5% Span, formulated in submicron particles using a microfluidizer).

• Cytokine inducing agents (see more detail below). Cytokine inducing agents suitable for use in the invention include toll-like receptor agonists (TLR7), (for example, the benzonaphthyridine compounds disclosed in 2009/111337.

• Saponins (Chapter 22 Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)), which are a heterologous group of sterols glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be obtained commercially from Smilax ornata (sarsaparilla), Gypsophilla paniculata (bridal veil), and Saponaria officinalis (soap dish). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOM. QS21 is marketed as STIMULON (TM). Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, which includes QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A production process of QS21 is disclosed in Patent No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (WO96 / 33739). Combinations of saponins and cholesterols can be used to form unique particles called immunostimulatory complexes (ISCOM) (Chapter 23 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (iSBN 0-306-44867-X) ). ISCOMs also normally include a phospholipid such as a phosphatidylethanolamine or phosphatidylcholine. Any known saponin in ISCOMs can be used. Preferably, ISCOMs include one or more of QuilA, QHA and QHC. ISCOMs are further described in WO96 / 337391, EP-A-0109942 and WO96 / 11711. Optionally, ISCOMs may be devoid of additional detergents (WO00 / 07621). A review of the development of saponin-based adjuvants can be found in Barr et al. (1998) Advanced Drug Delivery Reviews 32: 247-271 and Sjolanderet et al. (1998) Advanced Drug Delivery Reviews 32: 321-338. • Fatty adjuvants (see in more detail below), including oil-in-water emulsions, modified natural As lipids from enterobacterial lipopolysaccharides, phospholipid compounds (such as the synthetic phospholipid dimer, E6020) and the like.

• ADP ribosilant bacterial toxins (for example, thermally labile enterotoxin "LT" of E. coli, coli toxin "CT", or pertussis toxin "PT") and detoxified derivatives thereof, such as mutant toxins known as LT-K63 and LT-R72 (Pizza et al. (2000) Int J Med Microbiol 290: 455-461). The use of detoxified ADP ribosilant toxins as mucosal adjuvants is described in WO95 / 17211 and as parenteral adjuvants in WO98 / 42375.

• Bioadhesives and mucoadhesives, such as esterified hyaluronic acid microspheres (Singh et al (2001) J Cont Release 70: 267-276) or chitosan and its derivatives (WO99 / 27960).

• Microparticles ie a particle of ~ 100 nm to ~ 150 pm in diameter, more preferably, of ~ 200 nm to ~ 30 | jm in diameter or -500 nm to -10 jm in diameter) formed of materials that are biodegradable and non-toxic (for example, a poly (a-hydroxy acid), a polyhydroxybutyric acid, a polyortoester, a polyanhydride, a polycaprolactone, and the like), with the poly (lactide-co-glycolide) being preferred, optionally treated to have a charged surface negatively (for example, with SDS) or a positively charged surface (for example, with a cationic detergent, such as CTAB).

• Liposomes (Chapter 13 and 14 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)). Examples of liposome formulations suitable for use as adjuvants are described in U.S. Patent No. 6,090,406, U.S. Patent No. 5,916,588 and EP-A-0626169.

• Polyoxyethylene ethers and polyoxyethylene esters (WO99 / 52549). Such formulations include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO01 / 21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9 lauryl ether (laureth 9), polyoxyethylene-9 stearyl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene 4 lauryl ether, polyoxyethylene lauryl35, and lauryl ether35 polyoxyethylene ether 23.

• Muramyl peptides, such as N-acetylmuramyl-L-threonyl-D-isoglutamine ("thr-MDP"), N -acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylglucosaminyl-N -acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide ("DTP-DPP", or "Theramide ™), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2'dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy) -ethylamine ("MTP-PE").

• a proteasome preparation with an outer membrane protein prepared from a first Gram-negative bacterium in combination with a lipopolysaccharide preparation derived from a second Gram-negative bacterium, in which the proteasome with the outer membrane protein and the preparations of Lipopolysaccharides form a stable non-covalent adjuvant complex. Such complexes include "IVX-908", a complex comprised of an outer membrane of Neisseria meningitidis and lipopolysaccharides.

• A polyoxidonium polymer (Dyakonova et al. (2004) Int Immunopharmacol 4 (13): 1615-23, FR-2859633) or other N-oxidized polyethylene piperazine derivatives.

• 5'-Methyl inosine monophosphate ("MIMP") (Signorelli & Hadden (2003) Int Immunopharmacol 3 (8): 1177-86).

• A polyhydroxylated pyrrolizidine compound (W02004 / 064715), such as one having the formula:

where R is selected from the group comprising hydrogen, acyl, alkyl, (eg cycloalkyl), linear or branched, unsubstituted or substituted, saturated or unsaturated alkenyl, alkynyl, and aryl groups or a pharmaceutically acceptable salt thereof. Examples include, but are not limited to: casuarina, casuarina-6-a-D-glucopiranosa, 3-epi-casuarina, 7-epi-casuarina, 3,7-diepi-casuarina, and the like

• a CD1d ligand, such as an a-glycosylceramide (De Libero et al., (2005) Nature Reviews Immunology 5: 485-496; US Patent No. 5,936,076; Old et al., J Clin Investig, 113 : 1631-1640; US2005 / 0192248; Yang et al. (2004) Angew Chem Int Ed 43: 3818-3822; WO2005 / 102049; Goffet et al (2004) Am Chem Soc 126: 13602-13603; WO03 / 105769) (for example, a-galactosylceramide), a-glycosylceramides containing phytosphingosine, OCH, KRN7000 [(2S, 3S, 4R) -1-O- (aD-galactopyranosyl) -2- (N-hexacosanoylamino) -1, 3,4-octadecanotriol], CRONY-101, 3 "-O-sulfogalactosylceramide, etc.

• A gamma inulin (Cooper (1995) Pharm Biotechnol 6: 559-80) or a derivative thereof, such as algamulin.

• Virosomes and virus-like particles (VLP). These structures generally contain one or more proteins from an optionally combined virus or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the natural viral genomes. Viral proteins can be produced recombinantly or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), hepatitis B virus (such as core proteins or capsid), hepatitis E virus, Measles virus, Sindbis virus, Rotavirus, Foot and mouth disease virus, Retrovirus, Norwalk virus, human papillomavirus, HIV, RNA phages, Qp phage (such as protein coatings), GA phage, phage fr, phage AP205, and Ty (such as the p1 protein of the Ty retrotransposon).

These and other adjuvant active substances are described in more detail in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X) and Vaccine Adjuvants: Preparation Methods and Research Protocols ( Volume 42 of the Methods in Molecular Medicine series). ISBN: 1-59259 083-7. Ed. O'Hagan.

The compositions may include two, three, four or more adjuvants. For example, the compositions of the invention may advantageously include an oil-in-water emulsion and a cytokine inducing agent or a mineral-containing composition and a cytokine-inducing agent, or two oil-in-water emulsion adjuvants, or two benzonaphthyridine compounds, etc.

The antigens and adjuvants in a composition will normally be in premix.

Oily emulsion adjuvants

Compositions of oily emulsions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5% squalene, 0.5% Tween 80, and 0.5% Span 85, formulated in particles submicrometers using a microfluidizer). Freund's complete adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used.

Several oil-in-water emulsions are known, and typically include at least one oil and at least one surfactant, the oils and surfactants being biodegradable (metabolizable) and biocompatible. Droplets of Oil in the emulsion are generally less than 5 j in diameter, and can even have a submicron diameter, these small sizes being achieved with a microfluidizer to provide stable emulsions. Droplets smaller than 220 nm are preferred since they can be sterilized by filtration.

The invention can be used with oils such as those from an animal source (such as a fish) or a vegetable source. Sources of vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, illustrates nut oils. Jojoba oil can be used, for example, obtained from jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the group of grain oils, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, tritical and the like can also be used. The fatty acid esters of 6 10 carbon atoms of glycerol and 1,2-propanedyl, although not naturally occurring in seed oils, can be prepared by hydrolysis, separation and esterification of suitable materials starting from nut oils and seeds. Fats and oils of mammalian milk are metabolizable and can therefore be used in the practice of the present invention. The separation, purification, saponification and other means necessary to obtain pure oils from animal sources are well known in the art. Most fish contains metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti illustrates some of the fish oils that can be used herein. Numerous branched chain oils are biochemically synthesized in isoprene units of 5 carbon atoms and are generally referred to as terpenoids. Shark liver oil contains a branched unsaturated terpenoid known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane, which is particularly preferred herein. document. Squalane, the saturated analog of squalene, is also a preferred oil. Fish oils, including squalene and squalane, are readily available from commercial sources or can be obtained by methods known in the art. Other preferred oils are tocopherols (see below). Mixtures of oils can be used.

Surfactants can be classified by their 'HLB' (hydrophilic / lipophilic balance. Preferred surfactants of the invention have an HLB of at least 10, preferably at least 15, and more preferably at least 16. The invention can be used with surfactants including, but not limited to: polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) , marketed under the trade name DOWFAX (TM), such as linear EO / PO block copolymers; octoxyols, which may vary in the number of the repeating ethoxy groups (oxy-1,2-ethanediyl), being of particular interest octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol); (octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the TERGIT series OL (TM) NP; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Nonionic surfactants are preferred. Preferred surfactants to include in the emulsion are TWEEN 80 (TM) (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.

Surfactant mixtures may be used, for example, mixtures of TWEEN 80 (TM) / Span 85. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (TWEEN 80 (TM)) and an octoxynol such as t-octylphenoxy-polyethoxyethanol (Triton X-100). Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as polysorbate 80 (TM)) 0.01 to 1%, in particular about 0.1%; octyl or nonylphenoxy polyoxyethanes (such as Triton X-100, or other detergents of the Triton series) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to: a submicron emulsion of squalene, TWEEN 80 (TM), and Span 85. The volume emulsion composition may be approximately squalene to Approximately 5%, 0.5% polysorbate 80 and approximately 0.5% Span 85. In terms of weight, these ratios become squalene at 4.3%, polysorbate 80 at 0.5% and Span 85 at 0.48%. The present adjuvant is known as 'MF59' (documents WO90 / 14837; Podda & Del Giudice (2003) Expert Rev Vaccines 2: 197-203; Podda (2001) Vaccine 19: 2673-2680), as described in more detail in Chapter 10 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X) and Chapter 12 of Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of the series Methods in Molecular Medicine). ISBN: 1-59259-083-7. Ed. O'Hagan. The MF59 emulsion advantageously includes citrate ions, for example, 10 M sodium citrate buffer.

• An emulsion of squalene, a tocopherol, and TWEEN 80 (TM). The emulsion may include phosphate buffered saline. It can also include Span 85 (for example, 1%) and / or lecithin. These emulsions can have 2 to 10% squalene, 2 to 10% tocopherol and 0.3 to 3% TWEEN 80 (TM), and the weight ratio of squalene: tocopherol is preferably <1 since this provides a more stable emulsion. Squalene and TWEEN 80 / TM) may be present in a volumetric ratio of approximately 5: 2. One of said emulsions can be prepared by dissolving TWEEN 80 (TM) in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5g of DL-a-tocopherol and 5 ml of squalene), microfluidizing Then the mixture. The resulting emulsion may have submicron oil droplets, for example, with an average diameter between 100 and 250 nm, preferably about 180 nm.

• An emulsion of squalene, a tocopherol, and a Triton detergent (for example, Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer.

• An emulsion comprising a polysorbate (for example, polysorbate 80), a Triton detergent (for example, Triton X-100) and a tocopherol (for example, an a-tocopherol succinate). The emulsion can include these three components in a mass ratio of approximately 75:11:10 (for example, 750 pg / ml of polysorbate 80, 110 pg / ml of Triton X-100 and 100 pg / ml of a- succinate tocopherol), and these concentrations should include any contribution of these components from the antigens. The emulsion may also include squalene. The emulsion may also include a 3d-MPL (see below). The aqueous phase may contain a phosphate buffer.

• An emulsion of squalane, polysorbate 80 and poloxamer 401 ("PLURONIC (TM) L121"). The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful administration vehicle for muramyl dipeptides, and has been used with threonyl-MDP in adjuvant "SAF-1" (Allison & Byars (1992) Res Immunol 143: 519-25) (Thr-MDP at 0.05-1%, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without Thr-MDP, as in adjuvant "AF" (Hariharan et al. (1995) Cancer Res 55: 3486-9) (5% squalane, 1.25% Pluronic L121 and 80% polysorbate 0.2%) Microfluidization is preferred.

• An emulsion comprising squalene, an aqueous solvent, a hydrophilic nonionic alkyl polyoxyethylene ether surfactant (for example, polyoxyethylene ester ketostearyl ester (12) and a hydrophobic nonionic surfactant (for example, a sorbitan ester or manide ester, such as sorbitan monolaurate or 'Span 80') The emulsion is preferably heat-reversible and / or has at least 90% of the oil droplets (by volume) smaller than 200 nm in size. The emulsion may also include one or more of: alditol; a cryoprotective agent (for example, a sugar, such as dodecylmaltoside and / or sucrose); and / or an alipolyglycoside. Such emulsions may be lyophilized.

• An emulsion that has 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant. As described in reference WO95 / 11700, the preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron size droplets are advantageous.

• A submicron emulsion of oil in water of a non-metabolizable oil (such as a light mineral oil) and at least one surfactant (such as lecithin, TWEEN 80 (TM) or Span 80). Additives, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in US Patent No. 6,080,725, produced by the addition of an aliphatic amine, deacysaponin by means of glucuronic acid carboxyl group), dimethyldioctadecylammonium bromide and / or N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine.

• An emulsion comprising a mineral oil, a nonionic lipophilic ethoxylated fatty alcohol, and a hydrophilic nonionic surfactant (for example, an ethoxylated fatty alcohol and / or a polyoxyethylene polyoxypropylene block copolymer).

• An emulsion comprising a mineral oil, a nonionic hydrophilic ethoxylated fatty alcohol, and a hydrophilic nonionic surfactant (eg, an ethoxylated fatty alcohol and / or a polyoxyethylene polyoxypropylene block copolymer).

• An emulsion in which a saponin (for example, QuilA or QS21) and a sterol (for example, a cholesterol) are associated as helical micelles (WO2005 / 097181).

Emulsions can be mixed with antigen extemporaneously, at the time of administration. In this way, the adjuvant and the antigen can be kept separately in a distributed container or vaccine, ready for final formulation at the time of use. The antigen will generally be in aqueous form, so that the vaccine is finally prepared by mixing two liquids. The volume ratio of the two liquids to be mixed may vary (for example, between 5: 1 and 1: 5) but is generally about 1: 1.

Cytokine inducing agents

Cytokine inducing agents for inclusion in the compositions of the invention are capable, when administered to a patient, of stimulating the immune system to release cytokines, including interferons and interleukins. Preferred agents may induce the release of one or more of: interferon-Y; interleukin-1; interleukin-2; interleukin-12; TNF-a; TNF-p; and GM-CSF. Preferred agents that stimulate the release of cytokines associated with a Th1 type immune response, for example, interferon-y, TNF-a, interleukin-2. Stimulation of both interferon-Y and interleukin-2 is preferred.

As a result of receiving a composition of the invention, therefore, a patient may have T lymphocytes that, when stimulated with a VRS F protein, will release the one or more desired cytokines from a specific form of the antigen. For example, purified T lymphocytes from your blood will release Y-interferon when exposed to protein F in vitro. Methods for measuring such responses in peripheral blood mononuclear cells (PBMC) are known in the art, and include ELISA, ELISPOt , flow cytometry and real-time PCR. For example, Tassignon et al. (2005) J Immunol Meth 305: 188-98 report a study in which immune responses mediated by antigen-specific T lymphocytes against tetanus toxoid, specifically, yinterferon responses, were monitored, and it was discovered that the ELISPOT was the most sensitive procedure to discriminate antigen-specific T-cell-induced responses from spontaneous responses, but that the detection of intracytoplasmic cytokine by flow cytometry was the most effective procedure to detect the effects of restimulation.

Suitable cytokine inducing agents include, but are not limited to:

• an immunostimulatory oligonucleotide, such as one containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine) or a double stranded RNA, or an oligonucleotide containing a palindromic sequence, or an oligonucleotide which contains a poly (dG) sequence.

• Monophosphoryl lipid A 3-O-deacylated ('3dMPL', also known as "MPL (TM)") (Myers et al. (1990) pages

145-156 of Cellular and molecular aspects of endotoxin reactions; Ulrich (2000) Chapter 16 (pages 273-282) of Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of the Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan; Johnson et al. (1999) J Med Chem 42: 4640-9; Baldrick et al. (2002) Regulatory Toxicol Pharmacol 35: 398-413).

• An imidazoquinoline compound, such as IMIQUIMOD (TM) ("R-837") (United States Patent No. 4,680,338, United States Patent No. 4,988,815), RESIQUIMOD (TM) ("R- 848 ") (WO92 / 15582), and its analogues; and salts thereof (for example, hydrochloride salts). Other details concerning immunostimulatory imidazoquinolines can be found in Stanley (2002) Clin Exp Dermatol 27:57 1-577; Wu et al. (2004) Antiviral Res. 64 (2): 79-83; Vasilakos et al. (2000) Cell Immunol. 204 (1): 64-74; U.S. Patents Nos. 4689338, 4929624, 5238944, 5266575, 5268376, 5346905, 5352784, 5389640, 5395937, 5482936, 5494916, 5525612, 6083505, 6440992, 6627640, 6664264, 6664265, 66673126, 66773, 66773 , 6743920, 6800624, 6809203, 6888000, and 6924293; and Jones (2003) Curr Opin Investig Drugs

4: 214-218.

• A benzonaphthyridine compound, such as: (a) a compound having the formula:

in which:

R4 is selected from H, halogen, -C (O) OR7, -C (O) R7, -C (O) N (R11R12), -N (R11R12), -N (R9) ² , -NHN (R9) ^2, -SR 7, - (CH ²⁾ NOR7, - (CH ²⁾ n R 7, -LR8, -LR10, -OLR8, -OLR10, C ¹ -C ⁶ alkyl, heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkene , C ² -C ^8, C ² -C ⁸ alkyne, alkoxy C ¹ -C ⁶ haloalkoxy C ¹ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heterocycloalkyl and C ³ -C ^8, alkyl groups which ^C1 - ^C6 heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkene ^C2 - C8, C2-C8 alkyne, Ci-C6 alkoxy, haloalkoxy Ci-C6 aryl, heteroaryl, C3 -C8, and C3-C8 heterocycloalkyl of R4 are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, -NO2, -R7, -OR8, -C (O) R8, -OC ( O) R8, -C (O) OR8, -N (R9) 2, -P (O) (OR8) ² , -OP (O) (OR8) ² , -P (O) (OR10) ² , -OP (O) (OR10) ² , -C (O) N (R9) ² , -S (O) ² R8, -S (O) R8, -S (O) ² N (R9) ² , and -NR9S ( O) 2R8;

each L is independently selected from a bond, - (O (CH ²⁾ m) t-, C ¹ -C ⁶ alkylene , C ² -C ⁶ alkynylene , C ² -C ⁶ alkyl wherein said C ¹ - C ⁶ alkylene ^C2 - ^C6 alkynylene ² -C ⁶ C L are each optionally substituted with 1 to 4 substituents independently selected from halogen,

-R8, -OR8, -N (R9) ² , -P (O) (OR8) ² , -OP (O) (OR8) ² , -P (O) (OR10) ² , and -OP (O) ( OR10) ² ;

R7 is selected from H, C ¹ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C 6, C ² -C ⁸ alkene, alkyne , C ² -C ⁸ , alkoxy C ¹ -C ⁶ haloalkoxy C ¹ -C ⁶ heterocycloalkyl , C ³ -C ^8, alkyl groups which C1-C6 aryl, heteroaryl, C3-C8 cycloalkyl, C1-C6 heteroalkyl, C1-C6 , C2-C8 alkene, alkyne

C2-C8, C1-C6 alkoxy, C1-C6 haloalkoxy and C3-C8 heterocycloalkyl of R7 are each optionally substituted with 1 to 3 R13 groups; each R8 is independently selected from H, -CH (R10) 2, C1-C8 alkene , C ² -C ^8, C ² -C ⁸ alkyne, haloC ¹ -C ⁶ alkoxy C ¹ -C ⁶ heteroalkyl alkyl C ¹ -C ⁶ heterocycloalkyl C ² -C cicloal ⁸ hydroxyalkyl and ^C1 - ^C6 haloalkoxy ^C1 - ^C6), in which the alkyl groups C ¹ -C ⁸ alkene , C ² -C, C ² -C alkyne ⁸ heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkoxy C ¹ -C ⁶ cycloalkyl C ³ -C ⁸ heterocycloalkyl C ² -C 8 hydroxyalkyl, C1-C1-C6 haloalkoxy C6y R8 are each they optionally substituted with from 1 to

3 substituents independently selected from -CN, R11, -OR11, -SR11, -C (O) R11, -OC (O) R11, -C (O) N (R9) 2, -C (O) OR11, -NR9C (O) R11, -NR9R10, -NR11R12, -N (R9) 2, -OR9, -OR10, -C (O) NR11R12, -C (O) NR11OH, -S (O) ² R11, -S (O ) R11, -S (O) ² NR11R12, -NR11S (O) ² R11, -P (O) (OR11) ² , and -OP (O) (OR11) ² ;

each R9 is independently selected from H, -C (O) R8, -C (O) OR8, -C (O) R10, -C (O) OR10, -S (O) ² R10,

Ci-Ca alkyl, C ¹ -C ⁶ heteroalkyl and C ³ -C ⁶ cycloalkyl, or each R9 is independently a C ¹ -C ⁶ alkyl which together with the N to which they are attached form a C ³ -C ⁸ heterocycloalkyl, in which the heterocycloalkyl ring

C ³ -C ⁸ optionally containing a heteroatom selected option from N, O and S, and wherein the alkyl groups ^C1 - ^C6 heteroalkyl C ¹ -C ⁶ cycloalkyl or C ³ -C ⁶ heterocycloalkyl , C ³ -C ⁸ of R9 are each optionally substituted with 1 to 3 substituents independently selected from -CN, R11, -OR11, -SR11, -C (O) R11, -OC (O) R11, -C (O) OR11, -NR11R12, -C (O) NR11R12, -C (O) NR11OH, -S (O) 2R11, -S (O) R11, -S (O) ² NR11R12, -NR11S (O) ² R11, -P ( O) (OR11) ² , and -OP (O) (OR11) ² ;

each R10 is independently selected from aryl, cycloalkyl C ³ -C ⁸ heterocycloalkyl C ³ -Cs and heteroaryl, wherein aryl groups, cycloalkyl C ³ -C ⁸ heterocycloalkyl , C ³ -C ⁸ and heteroaryl are optionally substituted with 1 to 3 substituents selected from halogen, -R8, -OR8, -LR9, -LOR9, -N (R9) ² , -NR9C (O) R8, -NR9CO ² R8, -CO ² R8, -C (O) R8 and -C (O) N (R9) ² ;

R11 and R12 are independently selected from H, C ¹ -C ⁶ heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heterocycloalkyl and C ³ -C ^8, which C ¹ -C ⁶ alkyl groups heteroalkyl

C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heterocycloalkyl and C ³ -C ⁸ R11 and R12 are each optionally substituted with 1 to 3 substituents independently selected between halogen, -CN, R8, -OR8, -C (O) R8, -OC (O) R8, -C (O) OR8, -N (R9) ² , -NR8C (O) R8, -NR8C (O ) OR8, C ³ -C ⁸ heterocycloalkyl, -S (O ^ R8, -S (O) ^2N (R9) ^2, -NR 9S (O) ² R8, halo ^C1 - ^C6 haloalkoxy and C ¹ -C ⁶ ;

or R11 and R12 are each independently C ¹ -C ⁶ alkyl and taken together with the N atom to which they are attached form an optionally substituted C ³ -C ⁸ heterocycloalkyl ring containing an additional heteroatom selected from N, O and S;

Each R13 is independently selected from halogen, -CN, -LR9, -LOR9, -OLR9, -LR10, -LOR10, -OLR10,

-LR8, -LOR8, -OLR8, -LSR8, -LSR10, -LC (O) R8, -OLC (O) R8, -LC (O) OR8, -LC (O) R10, -LOC (O) OR8, -LC (O) NR9R11, -LC (O) NR9R8, -LN (R9) 2, -LNR9R8, -LNR9R10, -LC (O) N (R9) 2, -LS (O) 2R8, -LS (O) R8, -LC (O) NR8OH, -LNR9C (O) R8, -LNR9C (O) OR8, -LS (O) 2N (R9) 2, -OLS (O) 2N (R9) 2, -LNR9S (O) 2R8, -LC (O) NR9LN (R9) 2, -LP (O) (OR8) ² , -LOP (O) (OR8) ² , -LP (O) (OR10) ² and -OLP (O) (OR10 ) ² ;

each Ra is independently selected from -R8, -R7, -Or 7, -OR8, -R10, -OR10, -SR8, -NO ² , -CN, -N (R9) ² , -NR9C (O) R8, -NR9C (S) R8, -NR9C (O) N (R9) 2, -NR9C (S) N (R9) 2, -NR9CO2R8, -NR9NR9C (O) R8, -NR9NR9C (O) N (R9) 2, -NR9NR9CO2R8, -C (O) C (O) R8, -C (O) CH2C (O) R8, -CO2R8, - (CH2), CO2R8, -C (O) R8, -C (S) R8, - C (O) N (R9) 2, -C (S) N (R9) 2, -OC (O) N (R9) 2, -OC (O) R8, -C (O) N (OR8) R8, -C (NOR8) R8, -S (O) 2R8, -S (O) 3R8, -SO2N (R9) 2, -S (O) R8, -NR9SO2N (R9) 2, -NR9SO2R8, -P (O) (OR8) 2, -OP (O) (OR8) 2, -P (O) (OR10) 2, -OP (O) (OR10) 2, -N (OR8) R8, -CH = CHCO ² R8, - C (= NH) -N (R9) ² , and - (CH ² ) nNHC (O) R8; or two adjacent RA substituents in the Ring

A forms a 5-6 member ring that contains up to two heteroatoms as ring members;

n is, independently in each case, 0, 1, 2, 3, 4, 5, 6, 7 or 8; each m is independently selected between 1, 2, 3, 4, 5 and 6, and

t is 1,2, 3, 4, 5, 6, 7 or 8; (b) a compound having the formula:

in which:

R4 is selected from H, halogen, -C (O) OR7, -C (O) R7, -C (O) N (R11R12), -N (R11R12), -N (R9) ² , -NHN (R9) ^2, -SR 7, - (CH ²⁾ NOR7, - (CH ²⁾ n R 7, -LR8, -LR10, -OLR8, -OLR10, C ¹ -C ⁶ alkyl, heteroalkyl C ¹ -C ⁶ haloalkyl Cr Ca, alkene C ² -C ^8, C ² -C ⁸ alkyne, alkoxy C ¹ -C ⁶ haloalkoxy C ¹ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heterocycloalkyl and C ³ -C ^8, in which alkyl groups ^C1 - ^C6 heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkene C ² -C 8, C ² -C ⁸ alkyne, alkoxy C ¹ -C ⁶ haloalkoxy ^C1 - ^C6 aryl, heteroaryl , C ³ -C ⁸ cycloalkyl, and C ³ -C ⁸ heterocycloalkyl of R 4 are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, -NO ² , -R7, -OR8, -C (O) R8, -OC (O) R8, -C (O) OR8, -N (R9) ² , -P (O) (OR8) ² , -OP (O) (OR8) ² , -P (O ) (OR10) ² , -OP (O) (OR10) ² , -C (O) N (R9) ² , -S (O) ² R8, -S (O) R8, -S (O) ² N ( R9) ² , and -NR9S (O) 2R8;

each L is independently selected from a bond, - (O (CH ²⁾ m) t-, C ¹ -C ⁶ alkylene , C ² -C ⁶ alkynylene , C ² -C ⁶ alkyl wherein said C ¹ - C ⁶ alkylene ^C2 - ^C6 alkynylene ² -C ⁶ C L are each optionally substituted with 1 to 4 substituents independently selected from halogen,

-R8, -OR8, -N (R9) ² , -P (O) (OR8) ² , -OP (O) (OR8) ² , -P (O) (OR10) ² , and -OP (O) ( OR10) ² ;

R7 is selected from H, C ¹ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C 6, C ² -C ⁸ alkene, alkyne , C ² -C ⁸ , alkoxy C ¹ -C ⁶ haloalkoxy C ¹ -C ⁶ heterocycloalkyl , C ³ -C ^8, alkyl groups which ^C1 - ^C6 aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heteroalkyl C ¹ -C ⁶ , C ¹ -C ⁶ haloalkyl, C ² -C ⁸ alkene, alkyne

C ² -C ⁸ alkoxy C ¹ -C ⁶ haloalkoxy ^C1 - ^C6 heterocycloalkyl and C ³ -C ⁸ R7 are each optionally substituted with 1 to 3 R13 groups; each R8 is independently selected from H, -CH (R10) ^2, C ¹ -C 8 alkyl, C ² -C alkene, alkyne , C ² -C ⁸ haloalkyl C ¹ -C ⁶ alkoxy C ¹ -C ⁶ heteroalkyl C ¹ -C ⁶ cycloalkyl C ³ -C ⁸ heterocycloalkyl , C ² -C ⁸ hydroxyalkyl and ^C1 - ^C6 haloalkoxy ^C1 - ^C6), wherein the alkyl groups C ¹ -C ⁸ alkene , C ² -C alkyne C ² -C ⁸ heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkoxy C ¹ -C ⁶ cycloalkyl C ³ -C ⁸ heterocycloalkyl C ² -C 8 hydroxyalkyl, ^C1 - ^C6 haloalkoxy and C ¹ -C ⁶ of R8 are each optionally substituted with 1 to

3 substituents independently selected from -CN, R11, -o R11, -SR11, -C (O) R11, -OC (O) R11, -C (O) N (R9) 2, -C (O) OR11, - NR9C (O) R11, -NR9R10, -NR11R12, -N (R9) 2, -OR9, -OR10, -C (O) NR11R12, -C (O) NR11OH, -S (O) ² R11, -S ( O) R11, -S (O) ² NR11R12, -NR11S (O) ² R11, -P (O) (OR11) ² , and -OP (O) (OR11) ² ;

each R9 is independently selected from H, -C (O) R8, -C (O) OR8, -C (O) R10, -C (O) OR10, -S (O) ² R10, -C ¹ -C alkyl ⁶ , C ¹ -C ⁶ heteroalkyl and C ³ -C ⁶ cycloalkyl, or each R9 is independently a C ¹ -C ⁶ alkyl which together with the N to which they are attached form a C ³ -C ⁸ heterocycloalkyl, in which the heterocycloalkyl ring

C ³ -C ⁸ optionally containing a heteroatom selected option from N, O and S, and wherein the alkyl groups ^C1 - ^C6 heteroalkyl C ¹ -C ⁶ cycloalkyl or C ³ -C ⁶ heterocycloalkyl , C ³ -C ⁸ of R9 are each optionally substituted with 1 to 3 substituents independently selected from -CN, R11, -OR11, -SR11, -C (O) R11, -OC (O) R11, -C (O) OR11, -NR11R12, -C (O) NR11R12, -C (O) NR11OH, -S (O) 2R11, -S (O) R11, -S (O) ² NR11R12, -NR11S (O) ² R11, -P ( O) (OR11) ² , and -OP (O) (OR11) ² ; each R10 is independently selected from aryl, cycloalkyl C ³ -C ⁸ heterocycloalkyl C ³ -Cs and heteroaryl, wherein aryl groups, cycloalkyl C ³ -C 8 heterocycloalkyl, C ³ -C ⁸ and heteroaryl are optionally substituted with 1 to 3 substituents selected from halogen, -R8, -OR8, -LR9, -LOR9, -N (R9) ² , -NR9C (O) R8, -NR9CO ² R8, -CO ² R ⁸ , -C (O) R8 and -C (O) N (R9) 2;

R11 and R12 are independently selected from H, C ¹ -C ⁶ heteroalkyl C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heterocycloalkyl and C ³ -C ^8, which C ¹ -C ⁶ alkyl groups heteroalkyl

C ¹ -C ⁶ haloalkyl C ¹ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ heterocycloalkyl and C ³ -C ⁸ R11 and R12 are each optionally substituted with 1 to 3 substituents independently selected between halogen, -CN, R8, -OR8, -C (O) R8, -OC (O) R8, -C (O) OR8, -N (R9) ² , -NR8C (O) R8, -NR8C (O ) OR8, C ³ -C ⁸ heterocycloalkyl, -S (0 ^ R, -S (0 ^ N (R9) 2, -NR 9 S (O) ² R8, halo ^C1 - ^C6 haloalkoxy and ^C1 - ^C6;

or R11 and R12 are each independently C ¹ -C ⁶ alkyl and taken together with the N atom to which they are attached form an optionally substituted C ³ -C ⁸ heterocycloalkyl ring containing an additional heteroatom selected from N, O and S;

Each R13 is independently selected from halogen, -CN, -LR9, -LOR9, -OLR9, -LR10, -LOR10, -OLR10,

-LR8, -LOR8, -OLR8, -LSR8, -LSR10, -LC (O) R8, -OLC (O) R8, -LC (O) OR8, -LC (O) R10, -LOC (O) OR8, -LC (O) NR9R11, -LC (O) NR9R8, -LN (R9) 2, -LNR9R8, -LNR9R10, -LC (O) N (R9) 2, -LS (O) 2R8, -LS (O) R8, -LC (O) NR8OH, -LNR9C (O) R8, -LNR9C (O) OR8, -LS (O) 2N (R9) 2, -OLS (O) 2N (R9) 2, -LNR9S (O) 2R8, -LC (O) NR9LN (R9) 2, -LP (O) (OR8) ² , -LOP (O) (OR8) ² , -LP (O) (OR10) ² and -OLP (O) (OR10 ) ² ;

each Ra is independently selected from -R8, -R7, -Or 7, -OR8, -R10, -OR10, -SR8, -NO ² , -CN, -N (R9) ² , -NR9C (O) R8, -NR9C (S) R8, -NR9C (O) N (R9) 2, -NR9C (S) N (R9) 2, -NR9CO2R8, -NR9NR9C (O) R8, -NR9NR9C (O) N (R9) ² , -NR9NR9CO ² R8, -C (O) C (O) R8, -C (O) CH ² C (O) R8, -CO ² R ⁸ , - (CH ² ) nCO ² R8, -C (O) R8 , -C (S) R8, -C (O) N (R9) 2, -C (S) N (R9) 2, -OC (O) N (R9) 2, -OC (O) R8, -C (O) N (OR8) R8, -C (NOR8) R8, -S (O) 2R8, -S (O) 3R8, -SO2N (R9) 2, -S (O) R8, -NR9SO2N (R9) 2 , -NR9SO2R8, -P (O) (OR8) 2, -OP (O) (OR8) 2, -P (O) (OR10) 2, -OP (O) (OR10) 2, -N (OR8) R8 , -CH = CHCO ² R8, -C (= NH) -N (R9) ² , and - (CH ² ) nNHC (O) R8;

n is, independently in each case, 0, 1,2, 3, 4, 5, 6, 7 or 8;

each m is independently selected between 1, 2, 3, 4, 5 and 6, and

t is 1,2, 3, 4, 5, 6, 7 or 8; or (c) a pharmaceutically acceptable salt of any of (a) or (b).

Other benzonaphthyridine compounds, and processes for preparing benzonaphthyridine compounds, are described in US 2009/111337 and in International Patent Application No. PCT / US2010 // 047587.

A benzonaphthyridine compound, or a salt thereof, can be used by itself, or together with one or more additional compounds. For example, a benzonaphthyridine compound can be used together with an oil-in-water emulsion or a mineral oil-containing composition. In a particular embodiment, a benzonaphthyridine compound is used together with an oil-in-water emulsion (for example, a squalene-water emulsion, such as MF59) or a mineral-containing composition (for example, a mineral salt such as a salt of aluminum or a calcium salt).

A thiosemicarbazone compound, such as those disclosed in W02004 / 060308. The processes of formulation, manufacture and screening of active compounds are also described in document W02004 / 060308. Thiosemicarbazones are especially effective in stimulating human peripheral blood mononuclear cells for the production of cytokines, such as TNF-a.

A triptantrin compound, such as those disclosed in W02004 / 064759. The processes of formulation, manufacture and screening of active compounds are also described in document W02004 / 064759. Thiosemicarbazones are especially effective in stimulating human peripheral blood mononuclear cells for the production of cytokines, such as TNF-a. •

A nucleoside analog, such as: (a) Isatorabine (ANA-245; 7-thia-8-oxoguanosine):

and prodrugs thereof; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the compounds disclosed in U.S. Patent No. 6,924,271; US2005 / 0070556 and U.S. Patent No. 5,658,731; (f) a compound having the formula:

in which:

R ¹ and R ² are each independently H, halo, -NRaRb, -OH, alkoxy C ^1-6, C ^1-6 alkoxy substituted heterocyclyl, substituted heterocyclyl, aryl C ^6-10, C ^6-10 substituted aryl, C ^1.6 alkyl or substituted C ^1-6 alkyl;

R ³ is absent, H, C ¹ alkyl. ^{6, C1-6} alkyl, substituted aryl C ^6-10, C ^6-10 aryl substituted heterocyclyl, or substituted heterocyclyl;

R ⁴ and R ⁵ are each independently H, halo, heterocyclyl, substituted heterocyclyl, -C (O) -Rd, C ¹ alkyl. ^{6, C1-6} alkyl substituted, or joined together to form a 5 membered ring as in R ^4-5:

j j - X i

3 / ^ R8

"x 2 R4-5

Rg

getting the union in the links indicated by a ■ / wv

X ¹ and X ² are each independently N, C, O, or S;

Re is H, halo, -OH, C ¹ alkyl - ^6, alkenyl C ² - ⁶ alkynyl C ² - ^6, -OH, -NRaRb, - (CH ²⁾ nO-Rc, -O- (C ^{1. 6} ), -S (O) pRe, or -C (O) -Rd;

R ⁹ is H, alkyl C ^1-6, alkyl C ^1.6 substituted heterocyclyl, substituted heterocyclyl or Rga, where Rga is:

getting the union in the link indicated by a ■ / wv

R ¹⁰ and R ¹¹ are each independently H, halo, C ^1-6 alkoxy, substituted C ^1.6 alkoxy, -NRaRb, or -OH; each Ra and Rb is independently H, C ^1-6 alkyl, substituted C ^1.6 alkyl, -C (O) Rd, C ^6-10 aryl;

each Rc is independently H, phosphate, diphosphate, triphosphate, C ^1-6 alkyl or substituted C ^1.6 alkyl;

each Rd is independently H, halo, C ^1-6 alkyl, substituted C ^1.6 alkyl, C ^1-6 alkoxy, substituted C ^1.6 alkoxy, -NH ² , -NH (C ^1.6 alkyl), -NH (C ^1.6 substituted alkyl) , -N (C ^1-6 alkyl) ² , -N (C ^1.6 substituted alkyl) ² , C ^6-10 aryl, or heterocyclyl;

each Re is independently H, ^C1-6 alkyl, C ^1.6 alkyl substituted aryl , C ^6-10, C ^6-10 aryl substituted heterocyclyl, or substituted heterocyclyl;

each Rf is independently H, C ^1-6 alkyl, substituted C ^1.6 alkyl, -C (O) Rd, phosphate, diphosphate or triphosphate; each n is independently 0, 1, 2, or 3;

each p is independently 0, 1, or 2; or

or (g) a pharmaceutically acceptable salt of any of (a) to (f), a tautomer of any of (a) to (f), or a pharmaceutically acceptable salt of the tautomer.

• Loxoribine (7-allyl-8-oxoguanosine) (US Patent No. 5,011,828).

• The compounds disclosed in WO2004 / 87 153, which include: acylpiperazine compounds, indoledione compounds, tetrahydraisoquinoline compounds (THIQ), benzocyclodione compounds, aminoazavinyl compounds, aminobenzimidazole quinolinone compounds (ABIQ) (US Pat. No. 6,605,617, WO02 / 18383), hydraptalamide compounds, benzophenone compounds, isoxazole compounds, sterol compounds, quinazilinone compounds, pyrrole compounds (WO2004 / 018455), anthraquinone compounds, quinoxaline compounds , triazine compounds, pyrazalopyrimidine compounds, and benzazole compounds (WO03 / 082272).

• The compounds disclosed in WO2006 / 002422.

• An aminoalkylglucosaminide phosphate derivative, such as RC-529 (Johnson et al. (1999) Bioorg Med Chem Lett 9: 2273-2278, Evans et al. (2003) Expert Rev Vaccines 2: 219-229).

• A phosphazene, such as poly [di (carboxyphenoxy) phosphazene] ("PCPP") as described, for example, in Andrianov et al. (1998) Biomaterials 19: 109-115 and Payne et al. (1998) AdvDrug Delivery Review 31: 185-196.

• Small molecule immunopotentiators (SMIP) such as:

N2-methyl-1- (2-methylpropyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine N2, N2-dimethyl-1- (2-methylpropyl) -1H-imidazo [4,5 -c] quinoline-2,4-diamine

N2-ethyl-N2-methyl-1- (2-methylpropyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine

N2-methyl-1- (2-methylpropyl) -N2-propyl-1H-imidazo [4,5-c] quinoline-2,4-diamine

1- (2-methylpropyl) -N2-propyl-1H-imidazo [4,5-c] quinoline-2,4-diamine N2-butyl-1- (2-methylpropyl) -1H-imidazo [4,5-c ] quinoline-2,4-diamine

N2-butyl-N2-methyl-1- (2-methylpropyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine

N2-methyl-1- (2-methylpropyl) -N2-pentyl-1H-imidazo [4,5-c] quinoline-2,4-diamine

N2-methyl-1- (2-methylpropyl) -N2-prop-2-enyl-1H-imidazo [4,5-c] quinoline-2,4-diamine

1- (2-methylpropyl) -2 - [(phenylmethyl) thio] -1H-imidazo [4,5-c] quinolin-4-amine

1- (2-methylpropyl) -2- (propylthio) -1H-imidazo [4,5-c] quinolin-4-amine

2- [[4-amino-1- (2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl] (methyl) amino] ethanol

(methyl) amino] 2 - [[4-amino-1- (2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl] ethyl acetate

4-amino-1- (2-methylpropyl) -1,3-dihydro-2H-imidazo [4,5-c] quinolin-2-one

N2-butyl-1- (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine

N2-butyl-N2-methyl-1- (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine

N2-methyl-1- (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine

N2, N2-dimethyl-1- (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine

1- [4-amino-2- [methyl (propyl) amino] -1H-imidazo [4,5-c] quinolin-1-yl] -2-methylpropan-2-ol

1- [4-amino-2- (propylamino) -1H-imidazo [4,5-c] quinolin-1-yl] -2-methylpropan-2-ol

N4, N4-dibenzyl-1- (2-methoxy-2-methylpropyl) -N2-propyl-1H-imidazo [4,5-c] quinoline-2,4-diamine.

The cytokine inducing agents for use in the present invention may be modulators and / or agonists of the Toll-like receptors (TLR). For example, they may be agonists of one or more of the TLR1, TLR2, TLR3, TLR4, TLR7, TLR8 and / or TLR9 proteins. Preferred agents are TLR4 agonists (for example the modified natural As liquid derived from bacterial lipopolysaccharides, phospholipid compounds, such as the synthetic phospholipid dimer, E6020), TLR7 (for example, benzonaphthyridines, imidazoquinolines) and / or TLR9 (for example, CpG oligonucleotides). These agents are useful for activating innate immunity pathways.

The cytokine inducing agent can be added to the composition at various times during its production. For example, it may be within an antigen composition, and then this mixture can be added to an oil-in-water emulsion. Alternatively, it can be inside an oil-in-water emulsion, in which case, the agent can either be added to the emulsion components before emulsification, or it can be added to the emulsion after emulsification. Similarly, the agent can be coacervated within the droplets of the emulsion. The location and distribution of the cytokine inducing agent within the final composition will depend on its hydrophilic / lipophilic properties, for example, the agent can be located in the aqueous phase, in the oil phase, and / or in the oil interface. Water.

The cytokine inducing agent can be conjugated to an independent agent, such as an antigen (for example, CRM197). A general review of conjugation techniques for small molecules is provided in Thompson et al. (2003) Methods in Molecular Medicine 94: 255-266. Alternatively, adjuvants may be non-covalently associated with additional agents, such as by hydrophobic or ionic interactions.

Preferred cytokine inducing agents are (a) benzonaphthyridine compounds; (b) immunostimulatory oligonucleotides and (c) 3dMPL.

Immunostimulatory oligonucleotides may include nucleotide modifications / analogs such as phosphorothioate modifications and may be double stranded or (except RNA) single stranded. Kandimalla et al. (2003) Nucleic Acids Research 31: 2393-2400, documents WO02 / 26757 and WO99 / 62923, reveal possible substitutions of analogues, for example, the replacement of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further studied in Krieg (2003) Nature Medicine 9: 831-835; McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32: 179-185; WO98 / 40100; U.S. Patent No. 6,207,646; U.S. Patent No. 6,239,116 and U.S. Pat. No. 6,429,199. A CpG sequence can be directed to TLR9, such as the GTCGTT or TTCGTT motif (Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3): 654-658). The CpG sequence may be specific to induce a Th ¹ immune response, such as a CpG-A ODN (oligodeoxynucleotide), or it may be more specific to induce a B lymphocyte response, such as a CpG-B ODN. CpG-A and CpG-B ODN are analyzed in Blackwell et al. (2003) J Immunol 170: 4061-4068, Krieg (2002) Trends Immunol 23: 64-65; and WO01 / 95935. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed such that the 5 'end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can be joined at their 3 'ends to form "immunomers." See, for example, references Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3): 654-658; Kandimalla et al. (2003) BBRC 306: 948-953; Bhagat et al. (2003) BBRC 300: 853-861 and WO03 / 035836. A useful CpG adjuvant is CpG7909, also known as PROMUNE (TM) (Coley Pharmaceutical Group, Inc.).

Alternatively, or additionally, when using CpG sequences, TpG sequences can be used (WO01 / 22972). These oligonucleotides may be free of unmethylated CpG motifs.

The immunostimulatory oligodeoxynucleotide can be rich in pyrimidine. For example, it may comprise more than one consecutive thymidine nucleotide (for example, TTTT (SEQ ID NO: 17), as disclosed in WO01 / 22972), and / or it may have a nucleotide composition with> 25% of Thymidine (for example,>35%,>40%,>50%,>60%,> 80%, etc.). For example, it may comprise more than one consecutive cytosine nucleotide (for example, CCCC (SEQ ID NO: 18), as disclosed in WO01 / 22972), and / or it may have a nucleotide composition with> 25% of cytosine (for example,>35%,>40%,>50%,>60%,> 80%, etc.). These oligonucleotides may be free of unmethylated CpG motifs.

Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They can comprise less than ^{1 00} nucleotides.

3dMPL (also known as monophosphoryl lipid A 3 of -O-acylated or as 3-O-deacyl-4'-monophosphoryl lipid A) is an adjuvant in which position 3 of the reducing end of glucosamine in monophosphoryl lipid A is has deaciled. 3dMPL has been prepared from a Salmonella minnesota liver-free mutant and is chemically similar to lipid A, but lacks an acid-sensitive phosphoryl group and a base-sensitive acyl group. Activates monocyte / macrophage lineage cells and stimulates the release of several cytokines, which includes IL-1, IL-12, TNF-a and GM-CSF (see also Thompson et al. (2005) J Leukoc Biol 78: ' The low-toxicity versions of LPS, MPL® adjuvant and RC529, are efficient adjuvants for CD4 + T cells'). The preparation of 3dMPL was originally described in British patent application GB-A-22202 11.

3dMPL can take the form of a mixture of related molecules, diversified by their acylation (for example, they have 3, 4, 5 or ⁶ acylated chains, which can have different lengths). The two glucosamine monosaccharides (also known as 2-deoxy-2-amino-glucose) are N-acylated at the carbon atoms of position 2 (that is, at positions 2 and 2 '), and there is also O- acylation in the 3 'position. The group attached to carbon atoms 2 has the formula -NH-CO-CH ² -CR1R1 '. The group attached to the 2 'carbon atom has the formula -NH-CO-CH ² -CR2R2'. The group attached to the 3 'carbon atom has the formula -O-CO-CH ² -CR3R3'. A representative structure is:

The groups R1, R ² and R ³ are each independently - (CH ² VCH ^3. The value of n is preferably between ⁸ and 16, more preferably between 9 and 12, and most preferably 10.

The groups R1 ', R2' and R3 'can each be independently: (a) -H; (b) -OH; or (c) -O-CO-R4, where R ⁴ is one any of -H or - (CH ² ) m-CH ³ , wherein the value of m is preferably between 8 and 16, and is more preferably 10, 12 or 14. In position 2, m is preferably 14. In the 2 'position, m is preferably 10. In the 3' position, m is preferably 12. The R1 ', R2' and R3 'groups are therefore preferably O-acyl groups of dodecanoic acid, tetradecanoic acid or hexadecanoic acid .

When all R1 ', R2' and R3 'are -H then the 3dMPL only has 3 acylated chains (one in each of positions 2, 2' and 3 '). When only two of R1 ', R2' and R3 'are -H then the 3dMPL can have 4 acylated chains. When only one of R1 ', R2' and R3 'is -H then the 3dMPL can have 5 acylated chains. When none of R1 ', R2' and R3 'is -H then the 3dMPL can have 6 acylated chains. The 3dMPL adjuvant used in accordance with the invention may be a mixture of these forms, with 3 to 6 acylated chains, but it is preferred to include a 3dMPL with 6 acylated chains in the mixture and, in particular, to ensure that the form of Hexaacyl chain accounts for 10% by weight of the total 3dMPL, for example,> 20%,> 30%,> 40%,> 50% or more. It has been found that a 3dMPL with 6 acylated chains is the most active form as an adjuvant.

Thus, the most preferred form of 3dMPL for inclusion in compositions of the invention has the formula (IV), shown below.

When 3dMPL is used in the form of a mixture, then references to the amounts or concentrations of 3dMPL in the compositions of the invention refer to the combination of 3dMPL species in the mixture.

Under aqueous conditions, 3dMPL can form micellar aggregates or particles of different sizes (for example, with a diameter <150 nm or> 500 nm. Either or both of these can be used in the invention and it is best that the particles can be selected in a routine test Smaller particles (for example, small enough to produce a transparent aqueous suspension of 3dMPL) are preferred for use in accordance with the invention due to their greater activity (WO94 / 21292). they may have an average diameter of less than 220 nm, more preferably less than 200 nm or less than 150 nm or less than 120 nm, and may even have an average diameter of less than 100 nm. In most cases, however, the mean diameter shall not be less than 50 nm. These particles are small enough to be suitable for sterilization by filtration. The particle diameter can be evaluated by the technique Routine unique dynamic light scattering, which reveals an average particle diameter. When a particle is said to have a diameter of x nm, generally, there will be a distribution of particles around this average value, but at least 50% in number (for example,> 60%,> 70%,> 80%, > 90%, or more) of the particles will have a diameter in the range x + 25%.

3dMPL can be used advantageously together with an oil-in-water emulsion. Virtually all of the 3dMPL can be placed in the aqueous phase of the emulsion. The 3dMPL can be used by itself, or together with one or more additional compounds. For example, the use of 3dMPL together with the saponin QS21 (WO94 / 00153) (including an oil-in-water emulsion (WO95 / 17210)), with an immunostimulatory oligonucleotide, with both QS21 and an immunostimulatory oligonucleotide, with aluminum phosphate (WO96 / 26741), with aluminum hydroxide (WO93 / 19780), or with aluminum phosphate and aluminum hydroxide at the same time.

Fatty adjuvants

The fatty adjuvants that can be used with the invention include the oil-in-water emulsions described above, and also include, for example:

• A phospholipid compound of formula I, II or III, or a salt thereof:

as defined in WO03 / 011223, such as' ER 803058 ',' ER 803732 ',' ER 804053 ', ER 804058', 'ER 804059', 'ER 804442', 'ER 804680', 'ER 804764' , ER 803022 or 'ER 804057' (for example,

ER804057 is also called E6020. A phospholipid compound of formula I, II or III, or a salt thereof, can be used by itself, or together with one or more additional compounds. For example, a compound of formula I, II or III, can be used together with an oil-in-water emulsion or a mineral oil-containing composition. In a particular embodiment, E6020 is used together with an oil-in-water emulsion (for example, a squalene-water emulsion, such as MF59) or a composition containing a mineral (for example, a mineral salt such as a salt of aluminum or a calcium salt).

Escherichia coli lipid A derivatives such as OM-174 (described in Meraldi et al. (2003) Vaccine 21: 2485-249 1 and Pajak et al. (2003) Vaccine 21: 836-842).

A formulation of a cationic lipid and a colipid (usually neutral), such as aminopropyldimethylmiristolethyloxy propanaminium diphitanoylphosphatidylethanolamine bromide ("VAXFECTIN (TM)") or aminopropyldimethylbis-dodecyloxy-propanaminium dioleoiphosphyl dihydropylamine (R) -PYL-RIPA-RIP-diamipropylamine bromide. Formulations containing (+) - N- (3-aminopropyl) -N, N-dimethyl-2,3-bis (sin-9-tetradeceneyloxy) -1-propanaminium salts (U.S. Patent No. 6,586,409).

Monophosphoryl lipid A 3-O-deacylated (see above).

Lipid-containing compounds bound to a phosphate-containing main acyl structure, such as the TLR4 antagonist, E5564 (Wong et al. (2003) J Clin Pharmacol 43 (7): 735-42, US2005 / 0215517):

Lipopeptides (i.e., compounds comprising one or more fatty acid residues and two or more residues of amino acids), such as glycerylcysteine-based lipopeptides. Specific examples of said peptides include compounds of the following formula

wherein each of R1 and R2 represents a saturated or unsaturated, aliphatic or mixed aliphatic-cycloaliphatic hydrocarbon radical having from 8 to 30, preferably from 11 to 21, carbon atoms that are also optionally substituted by oxygen functions, R3 represents hydrogen or the radical R ¹ -CO-O-CH ² - in which R1 has the same meaning as above, and X represents an amino acid linked by a peptide bond having a free, esterified or amidated carboxy group, or a amino acid sequence of 2 to 10 amino acids in which the carboxyl end group is in its free, esterified or amidated form. In certain embodiments, the amino acid sequence comprises an amino acid D, for example, D-glutamic acid (D-Glu) or D-gamma-carboxyglutamic acid (D-Gla).

Bacterial lipopeptides generally recognize TLR2, without the need for TLR6 to participate. (TLRs act cooperatively to provide specific recognition of different triggers, and TLR2 together with TLR6 recognize peptidoglycans, while TLR2 recognizes lipopeptides without TLR6). These are sometimes classified as natural lipopeptides and synthetic lipopeptides. Synthetic lipopeptides tend to behave similarly, and are recognized primarily by TLR2.

Lipopeptides suitable for use as adjuvants include compounds having the formula:

where the chiral scepter marked with * and marked with * * * are both in the R configuration;

the chiral center marked with ** is in any of the R or S configuration;

each R1 and R1bes independently an aliphatic or cycloaliphatic-aliphatic hydrocarbon group having 7-21 carbon atoms, optionally substituted by oxygen functions, or one of R1a and R1b, but not both, is H;

R2 is an aliphatic or cycloaliphatic hydrocarbon group having 1-21 carbon atoms and optionally substituted by oxygen functions;

n is 0 or 1;

As represents one of -O-Kw-CO- or -NH-Kw-CO-, where Kw is an aliphatic hydrocarbon group having 1-12 carbon atoms;

As1 is a D or L-alpha-amino acid;

each of Z1 and Z2 independently represents -OH or the N-terminus radical of a D or L alpha-amino acid of an amino- (lower alkane) -sulfonic acid or of a peptide having up to 6 amino acids selected from D and L-alpha aminocarboxylic acids and amino-lower alkyl sulfonic acids; Y

Z3 is H or -CO-Z4, where Z4 is -OH or the N-terminal radical of an alpha-amino acid D (L) -sulfonic acid or sulfide acid or a peptide having up to 6 amino acids selected from D and L-alpha aminocarboxylic acids and amino-lower alkyl sulfonic acids; or an ester or amide formed from the carboxylic acid of said compounds. Suitable amides include -NH ² and NH (lower alkyl), and suitable esters include C1-C4 alkyl esters, (lower alkyl or lower alkane, as used herein, refers to Ci-C6 chain alkyls linear or branched).

Such compounds are described in more detail in US 4,666,886. In a particular embodiment, the lipopeptide has the formula:

Another example of a species of lipopeptide is called LP40, and is a TLR2 agonist. Akdis, et al., Eur. J. Immunology, 33: 2717-26 (2003}.

It is related to a known class of E. coli- derived lipopeptides , referred to as murein lipoproteins. Some partial degradation products of these proteins called murein lipopeptides are described in Hantke, et al., Eur. J. Biochem., 34: 284-296 (1973). They comprise a peptide bound to an N-acetyl murmic acid and are therefore related to muramyl peptides, which are described in Baschang, et al., Tetrahedron, 45 (20): 6331-6360 (1989).

Aluminum salt adjuvants

Adjuvants known as "aluminum hydroxide" and "aluminum phosphate" can be used. These names are conventional, but are used only for convenience of use, since none of them is a precise description of the actual chemical compound that is present (for example, see Chapter 9 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)). The invention can use any of the "hydroxide" or "phosphate" adjuvants that are generally used as adjuvants.

Adjuvants known as "aluminum hydroxide" are typically aluminum oxyhydroxide salts, which are usually at least partially crystalline. Aluminum oxyhydroxide, which can be represented by the formula AIO (OH), can be distinguished from other aluminum compounds, such as aluminum hydroxide Al (OH) ³ , by infrared (IR) spectroscopy, especially by the presence of an absorption band at 1070 cm-1 and an intense shoulder at 3090-3100 cm-1 (Chapter 9 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867- X)). The degree of crystallinity of an aluminum hydroxide adjuvant is reflected by the half-height diffraction bandwidth (WHH), where the little crystalline particles show a greater line width due to smaller crystallite sizes. The surface area increases when the WHH does, and it has been observed that adjuvants with higher WHH values have greater capacity for antigen absorption. A fibrous morphology (for example, as seen in electronically transmitted micrographs) is typical of aluminum hydroxide adjuvants. The pI of the aluminum hydroxide adjuvants is typically about 11, that is, the adjuvant itself has a positive surface charge at physiological pH. Adsorption capacity between 1.8-2.6mg proteins per mg of Al +++ at pH 7.4 has been reported for aluminum hydroxide adjuvants.

Adjuvants known as "aluminum phosphate" are typically aluminum hydroxyphosphates, which often also contain a small amount of sulfate (ie, aluminum sulfate hydroxyphosphates). They can be obtained by precipitation, and reaction conditions and concentrations during precipitation affect the degree of hydroxyl substitution in the salt. Hydroxyphosphates generally have a PO ⁴ / Al molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AlPO ⁴ by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm-1 (for example, when heated to 200 ° C) indicates the presence of structural hydroxyls (Chapter 9 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman ) Plenum Press 1995 (ISBN 0-306-44867-X))

The molar ratio PO ⁴ / Al3 + of an aluminum phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95 + 0.1. Aluminum phosphate will generally be amorphous, especially for hydroxyphosphate salts. A typical adjuvant is an amorphous aluminum hydroxyphosphate with a molar ratio of PO ^ Al between 0.84 and 0.92, including 0.6 mg Al3 + / ml. Aluminum phosphate will generally be in the form of particles (for example, plate morphology as seen in electronically transmitted micrographs). Typical particle diameters are in the range of 0.5-20 pm (for example, approximately 5-10 pm) after adsorption of any antigen. Adsorption capacity between 0.7-1.5 mg proteins per mg of Al +++ at pH 7.4 has been reported for aluminum phosphate adjuvants.

The zero charge point (PZC) of aluminum phosphate is inversely related to the degree of hydroxyl phosphate substitution, and this degree of substitution may vary depending on the reaction conditions and the concentration of the reagents used to prepare the salt. by precipitation The PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate = pZc more acid) or by adding a buffer such as a histidine buffer (makes the PZC more basic). The aluminum phosphates used in accordance with the invention will generally have a PZC between 4.0 and 7.0, more preferably between 5.0 and 6.5, for example, approximately 5.7.

The aluminum salt suspensions used to prepare compositions of the invention may contain a buffer (for example, a phosphate or histidine or Tris buffer), but this is not always necessary. The suspensions are preferably sterile and free of pyrogens. A suspension may include free aqueous phosphate ions (for example, present at a concentration between 1.0 and 20 M, preferably between 5 and 15 M, and more preferably about 10 M. The suspensions may also comprise sodium chloride.

The invention can use a mixture of both aluminum hydroxide and aluminum phosphate. In this case, there may be more aluminum phosphate than hydroxide (for example, a weight ratio of at least 2: 1 (for example,> 5: 1,> 6: 1,> 7: 1,> 8: 1, > 9: 1, etc.

The concentration of Al +++ in a composition for administration to a patient is preferably less than 10 mg / ml for example, <5 mg / ml, <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. A preferred range is between 0.3 and 1 mg / ml. A maximum dose of 0.85 mg / dose is preferred.

Such as the inclusion of one or more aluminum salt adjuvants, the adjuvant component may include one or more additional adjuvants or immunostimulatory agents. Such additional components include, but are not limited to: a benzonaphthyridine compound, a monophosphoryl lipid A 3-O-deacylated adjuvant ('3d-MPL'); and / or an oil-in-water emulsion. 3d-MPL has also been referred to as monophosphoryl lipid A 3 de-O-acylated or as 3-O-deacyl-4'-monophosphoryl lipid A. The name indicates that position 3 of the reducing end of glucosamine in monophosphoryl lipid A is deaciled It has been prepared from a liver- free mutant of S.minnesota, and is chemically similar to lipid A, but lacks an acid-sensitive phosphoryl group and a base-sensitive acyl group. Activates monocyte / macrophage lineage cells and stimulates the release of several cytokines, which includes IL-1, IL-12, TNF-a and GM-CSF. The preparation of the 3d-MPL was originally described in British patent application GB-A-2220211, and the product is manufactured and sold by Corixa Corporation under the name MPL (TM). Other details can be found in Myers et al. (1990) pages 145-156 of Cellular and molecular aspects of endotoxin reactions; Ulrich (2000) Chapter 16 (pages 273-282) of Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of the Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan; Johnson et al. (1999) J Med Chem 42: 4640-9; and Baldrick et al. (2002) Regulatory Toxicol Pharmacol 35: 398-413.

The use of an aluminum hydroxide and / or aluminum phosphate adjuvant is useful, especially in children, and the antigens are usually adsorbed on these salts. Water squalene emulsions are also preferred, especially in the elderly. Useful adjuvant combinations include combinations of Th1 and Th2 adjuvants, such as CpG and alum, or resiquimod and alum. A combination of aluminum phosphate and 3dMPL can be used. Other combinations that can be used include: alum and a benzonapyridine compound or a SMIP, a squalene emulsion in water (such as MF59) and a benzonapyridine compound or a SMIP, and E6020 and a squalene emulsion in water, such as MF59) or alum.

The compositions of the invention can trigger a cell-mediated immune response, as well as a humoral immune response.

It is believed that two types of T lymphocytes, CD4 and CD8, are usually necessary to initiate and / or improve cell-mediated immunity and humoral immunity. CD8 T lymphocytes can express a CD8 correceptor and are commonly referred to as cytotoxic T lymphocytes (CTL). CD8 T lymphocytes are capable of recognizing or interacting with antigens presented in MHC class I molecules.

CD4 T lymphocytes can express a CD4 correceptor and are commonly referred to as helper T lymphocytes. CD4 T lymphocytes are capable of recognizing antigenic peptides bound to MHC class II molecules. After interaction with a class II molecule of m Hc, CD4 cells can secrete factors such as cytokines. These secreted cytokines can activate B lymphocytes, cytotoxic T lymphocytes, macrophages and other cells that participate in an immune response. Collaborative T lymphocytes or CD4 + cells can be further divided into two functionally distinct subsets: the TH1 phenotype and the H2 t phenotypes, which differ in their effector and cytokine function.

Activated TH1 lymphocytes enhance cellular immunity (including an increase in the production of antigen-specific CTLs) and therefore, are particularly valuable in the response to intracellular infections. TH1 lymphocytes can secrete one or more of IL-2, IFN-y and TNF-p. A TH1 immune response may result in local inflammatory reactions due to the activation of macrophages, NK lymphocytes (natural cytolytics) and cytotoxic CD8 T lymphocytes (CTL). A TH1 immune response can also act to expand the immune response by stimulating the growth of B and T lymphocytes with IL-12. B lymphocytes stimulated by TH1 can secrete IgG2a.

A TH1 immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH1 immune response (such as IL-2, IFN-y and TNF-p), an increase in activated macrophages, an increase in NK activity or an increase in the production of IgG2a. Preferably, the enhanced TH1 immune response will include an increase in the production of IgG2a.

A TH1 immune response can be triggered using a TH1 adjuvant. Generally, a TH1 adjuvant will cause increased levels of IgG2a production in relation to immunization of the antigen without adjuvant. TH1 adjuvants suitable for use in the invention may include, for example, formulations of saponin, virosomes and pseudoviral particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides. Immunostimulatory oligonucleotides, such as oligonucleotides containing a CpG motif, are preferred TH1 adjuvants for use in the invention.

The enhanced immune response may be one or both of a systemic and a mucosal immune response. Preferably, the immune response enables one or both of an improved systemic immune response and an improved mucosal.

Treatment, use and administration procedures

The compositions disclosed herein are suitable for administration to pregnant women and infants, in a method for inducing an immune response in a pregnant woman and / or baby, which comprises the step of administering a composition (eg, an inductive composition of an immune response directed against RSV) to the pregnant woman and / or baby. The compositions (for example, an inductive composition of an immune response directed against RSV) can be used to produce a vaccine formulation to immunize a pregnant woman and / or a baby. For example, the immune response can be triggered after administration of a purified VRS F protein, an alphavirus replicon particle, or a self-replicating RNA.

The invention also provides a composition of the invention for use as a medicament, for example, for use in immunizing a patient against RSV infection.

An inductive composition of an immune response directed against RSV is also described for use in the provision of a protective immunity against RSV in a baby by a method comprising (a) administering a first inductive composition of an immune response directed against the infant. RSV to a woman during pregnancy; and (b) administer a second inductive composition of an immune response directed against RSV to the baby born from pregnancy.

An inductive composition of an immune response directed against RSV is also described for use in triggering an immune response in a pregnant woman by a method comprising (a) administering a first inductive composition of an immune response directed against v Rs to the woman; and (b) administer a second inductive composition of an immune response directed against RSV to the baby born from pregnancy.

An inductive composition of an immune response directed against RSV is also described for use in the provision of a protective immunity against RSV in a baby, in which the baby was born from a woman to whom an inductive composition of a baby was administered. immune response directed against RSV during the time the woman was pregnant with the baby.

An inductive composition of an immune response directed against RSV is also described for use in the provision of a protective immunity against RSV in a baby, in which the baby has maternal antibodies against RSV.

The presence of maternal antibodies against RSV in the baby can be evaluated using any suitable procedure. For example, since the baby's immune system is immature, antibodies against RSV, especially IgG antibodies, present in the baby during the first weeks and months after birth (for example, from birth to about 2 or 3 or 4 months) are the maternal antibodies. Many procedures are known in the art to determine if there are maternal antibodies that bind to pathogen antigens. See, for example, Pappaioanou M, et al., Accurate Detection of Maternal Antibodies to HIV in Newborn Whole Blood Dried on Filter Paper, AIDS 7 (4): 483-488 (1993), and Pinon J.M. et al., Strategy for Diagnosis of Congenital Toxoplasmosis: Evaluation of Methods Comparing Mothers and Newborns and Standard Methods for Postnatal Detection of Immunoglobin G, M, and A Antibodies, J. Clin. Microbiol 39 (6): 2267-2271 (2001).

The use of a polypeptide as described above in the manufacture of a medicament to trigger an immune response in a patient is also described.

The immune response produced according to these procedures and use will generally include an antibody response, preferably a protective antibody response. Procedures for evaluating antibody responses after vaccination against RSV are well known in the art.

The compositions of the invention can be administered in numerous ways, such as intramuscular injection (eg, in the thigh, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and the like. The appropriate route of administration it will depend on the age, health status and other characteristics of the pregnant woman or the baby.A clinical specialist may determine an appropriate route of administration based on these and other factors.

The treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. In a multiple dose schedule, multiple doses may be administered by the same route or by different routes, for example, parenteral priming and mucosal reinforcement, mucosal stimulation and parenteral reinforcement, etc. The administration of more than one dose (typically two doses) is especially useful in patients without prior immune exposure. Multiple doses will typically be administered at intervals of at least 1 week (for example, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 6 weeks, approximately 8 weeks, approximately 10 weeks, approximately 12 weeks, approximately 16 weeks, and the like).

Vaccine formulations produced using a composition of the invention can be administered to patients at substantially the same time (for example, during the same doctor's visit or visit a healthcare professional or vaccination center) as other vaccines.

Examples

Example 1.

The efficacy and immunogenicity of the immunization regimen described herein can be evaluated, for example, using suitable animal models.

In one example, female cotton rats will receive an inductive composition of an immune response directed against RSV, before and / or after pairing to induce the production of antibodies against RSV during pregnancy. Alternatively, female cotton rats can become infected with RSV before pairing and receive a booster with an immune response inducing composition before and / or after pairing to increase the production of antibodies against RSV during pregnancy. The presence and titer of antibodies directed against RSV in the pregnant cotton rat can be evaluated using any suitable procedure, such as the neutralizing assays described herein or by an ELISA. Antibodies produced in the pregnant cotton rat will cross the placenta and enter the fetal circulation. After birth, neonatal rats will be breastfed by their mother or by a cotton rat not exposed to treatment (which has not been exposed to RSV or to an inductive composition of an immune response directed against RSV). Antibodies directed against RSV can be transferred to cotton rat pups by breastfeeding of females that were immunized with an inductive composition of an immune response directed against RSV or infected with RSV and reinforced with an inductive composition directed against RSV. Some of the newborn rats will receive an inductive composition of an immune response directed against RSV one or more times (for example, a stimulus followed by one or more reinforcements). When an inductive composition of an immune response directed against RSV is administered to a newborn two or more times, the compositions will be the same or different. Antibody levels directed against RSV in cotton rat pups can be evaluated at various time points after birth by any suitable procedure, such as neutralizing assays described herein or by an ELISA. The levels of protection against RSV transmitted by immunization regimens can be assessed by stimulating cotton rat pups with RSV and by evaluating virus titers in nasal washes or lungs of cotton rat pups or by examining the pathology of lungs or nasal cavity of cotton rat pups. Comparing the level of antibodies directed against RSV and the degrees of protection against RSV at different time points in offspring of cotton rats that have been actively immunized or not and who were born to mothers or breastfed females who had had different exposures to RSV (for example, none, RSV infection, immunization with an inductive composition of an immune response directed against RSV, or RSV infection followed by reinforcement with an inductive composition of an immune response directed against RSV) will allow evaluation of the efficacy and immunogenicity of the RSV immunization regimen described herein. In another example, the potential for low transplacental transfer efficiency of maternal antibodies to the fetus is resolved by manual transfer of preparations containing the antibody obtained from an adult cotton rat. An adult cotton rat will receive an inductive composition of an immune response directed against RSV to induce the production of antibodies directed against RSV or can be infected with RSV and receive a boost with an inductive composition of an immune response directed against RSV. Antibodies directed against RSV (or serum containing the antibodies) will be obtained from the cotton rat and administered to cotton rats not exposed to treatment (newborn or young cotton rats), for example, by intraperitoneal injection. Some of the cotton rats not exposed to the treatment will receive an inductive composition of an immune response directed against RSV one or more times (for example, a stimulus followed by one or more reinforcements). When an inductive composition of an immune response directed against RSV is administered cotton rats not exposed to the treatment two or more times, the compositions will be the same or different. Antibody levels directed against RSV in cotton rat pups can be evaluated at various time points after birth by any suitable procedure, such as neutralizing assays described herein or by an ELISA. The levels of protection against RSV transmitted by immunization regimens can be assessed by stimulating cotton rat pups with RSV and by evaluating virus titers in nasal washes or lungs of cotton rat pups or by examining the pathology of lungs or nasal cavity of cotton rat pups. Comparing the level of antibodies directed against v Rs and the degrees of protection against RSV at different time points in offspring of cotton rats that have been actively immunized or not and who received preparations containing antibodies obtained from cotton rats that had had different exposures to RSV (for example, none, RSV infection, immunization with an inductive composition of an immune response directed against RSV, or RSV infection followed by reinforcement with an inductive composition of an immune response directed against RSV ) will allow the evaluation of the efficacy and immunogenicity of the RSV immunization regimen described herein.

RSV neutralization test

HEp-2 cells were collected from confluent layers by trypsinization, resuspended in cell culture medium containing 5% bovine fetal serum (FBS) at a density of 6.5 x 10 6 cells / ml, seeded in 96 plates wells at 0.1 ml per well, and incubated at 37 ° C in 5% CO2 for 3-6 hours.

The test serum and control serum were thermally inactivated at 56 ° C for 30 minutes and serially diluted twice in phosphate buffered saline serum (PBS), 5% bovine serum albumin (BSA).

Serially diluted serum will be mixed with equal volumes of RSV that had been prediluted in 5% FBS in PBS to a concentration such that the added volume produces 80-140 syncytiums / well in the absence of test serum. Mixtures of RSV and serial diluted serum will be incubated for 2 hours at 37 ° C in 5% CO2.

The medium will be aspirated from the test plate seeded with HEp-2 and the virus-serum mixtures will be added to the wells of the plates. The plate will be incubated for 2 hours at 37 ° C in 5% CO ² . The virus-serum mixtures will be aspirated from the plate. A solution at room temperature of 0.75% methylcellulose in EMEM (free of phenol red), 5% FBS, will be added to the wells. The plate will then be incubated at 37 ° C in 5% CO ² for 40 hours.

The methylcellulose will be aspirated from the wells and 10% buffered formalin will be added to the wells. The plate will be incubated at room temperature for 1 hour. The plates will be washed three times with wash buffer (1 x PBS, 0.05% Tween 20). Permeabilization / blocking buffer (D-PBS, 2.5% FBS, 0.5% saponin, 0.1% NaAzide) will be added to the wells, and the plate will be incubated for 1 h at room temperature.

The permeabilization / blocking buffer will be aspirated from the wells. Then, the wells will be washed three times with wash buffer. A 1: 1000 dilution in permeabilization buffer / blocking monoclonal antibodies that recognize F and / or NP will be added to the wells. The plate will be incubated for 1 hour at room temperature.

The antibody solution will be aspirated. The wells will be washed three times with wash buffer. A 1: 1000 dilution in permeabilization buffer / blocking of a secondary antibody (goat antibody directed against mouse IgG conjugated with horseradish peroxidase and light chain) will be added to the wells. The plate will be incubated for 1 hour at room temperature.

The secondary antibody will be aspirated from the wells. The wells will be washed three times with wash buffer. The trypan blue substrate will be added to the wells. The plate will be incubated for 10 minutes at room temperature. The plate will be washed once with distilled water and aspirated to dryness.

The dyed syncytia will be counted using a UV analyzer with an image acquisition and counting software.

The syncytia will be counted in control wells that will have been incubated with viruses that would not have been mixed with serum. The syncytia will be counted in the sample wells. Two replicas of each well will be counted and averaged. The proportion of syncytia in the control wells that remain in the sample wells will be calculated. Serum dilution values that provide 60% virus neutralization are calculated by linear regression. The inverse of this dilution is the 60% neutralization titer.

Example 2: The VRS F subunit vaccine strengthens the response of RSV-specific antibodies in cotton rats and HIV-infected mice

1. Procedures

Subunit vaccine with the VRS F trimer

The VRS F trimer is a recombinant protein that comprises the ectodomain of VRS F with a deletion in the region of the fusion peptide that prevents association with other trimers. The resulting construction forms a homogeneous trimer, as observed by size exclusion chromatography, and has an expected phenotype consistent with a post-fusion conformation as observed by electron microscopy. The protein was expressed and purified in CHO cells. The resulting protein sample showed a purity greater than 95%. For the in vivo evaluation of the F subunit vaccine with aluminum hydroxide (alum), the F subunit was adsorbed on 2 mg / ml alum using 10 M histidine buffer pH 6.3, adjusted for isotonicity with sodium chloride. Subunit F was adsorbed onto alum overnight with gentle agitation at 2-8 ° C. For the duration of the FF subunit vaccine with MF59, the protein was diluted in PBS and mixed with an equal volume of 2X MF59 for 2 hours before immunization and mixed by gentle inversion.

Vaccination and infection of cotton rats and mice

Female cotton rats ( Sigmodon hispidis) were obtained from Harlan Laboratories and female BALB / c mice were obtained from Charles River Laboratories. All studies were approved and performed according to the Novartis Animal Care and Use Committee. Groups of 7 cotton rats were inoculated intranasally (in) with 1x105 plaque forming units (pfu) of RSV at 100 µl and were immunized intramuscularly (im) with the VRS F subunit vaccine (100 µl total volume at a posterior limb) 7 weeks later. Groups of 7 mice were inoculated with 1x106 pfu of RSV in 50 jl and im immunized with the VRS F subunit vaccine (100 µl of total volume divided between the two hind limbs) 11 and 15 weeks later. Two animals not exposed to the treatment were included in the study. Serum samples were collected at various time points after inoculation with RSV and 2 weeks after each immunization with the subunit of RSV F.

VRS F specific ELISA

Individual cotton or mouse rat serum samples were analyzed for the presence of RSV-specific IgG in an adsorption enzyme immunoassay (ELISA). ELISA plates (MaxiSorp 96 wells, Nunc) were coated overnight at 4 ° C with 1 jg / ml of purified RSV F protein in PBS. After washing (PBS with 0.1% Tween 20), the plates were blocked with Superblock blocking buffer in PBS (Thermo Scientific) for at least 1 h at 37 ° C. The plates were then washed, 5-fold serial dilutions of the serum in test diluent (PBS with 0.1% Tween 20 and 5% goat serum) from experimental or control animals were added, and the plates were incubated for 2 h at 37 ° C. After washing, the plates were incubated with a detection antibody against horseradish peroxidase-conjugated IgG (HRP) diluted in test diluent (1: 50,000 dilution of chicken antibody directed against cotton rat IgG [Immunology Consultants Laboratory, Inc,] or 1: 30,000 dilution of goat antibody directed against mouse IgG [Southern Biotech]) for 1 h at 37 ° C. Finally, the plates were washed and 100 µl of TMB peroxidase substrate solution (Kirkegaard & Perry Laboratories, Inc) was added to each well. The reactions were stopped by adding 100 µl of 1M H ³ PO ⁴ , and the absorbance was read at 450 nm using a plate reader. For each serum sample, a graphical representation of the optical density (OD) was generated against the logarithm of the inverse of the serum dilution by non-linear regression (GraphPad Prism). Titers were defined as the reciprocal of the serum dilution at OD ⁴⁵⁰ nm of about ^{0, 6,} normalized to a standard serum sample included on each plate (standard rat cotton - pooled serum of rats cotton infected with RSV with a defined titer of 1: 2500, mouse pattern - combined serum of BALB / c mice vaccinated with the VRS F subunit with adjuvant with a defined titer of 1: 269,773). If the titer of an individual sample is below the first serum dilution analyzed, 1:25, a 1: 5 titer was assigned for the purpose of calculating the group mean.

RSV neutralization test

Serum samples were analyzed to determine the presence of RSV neutralizing antibodies by means of a neutralization assay of spot reduction. Twice serial dilutions of thermally inactivated serum (HI) (in PBS with 5% HI-FBS) were added to an equal volume of RSV Long previously titrated to give approximately 115 pfu / 25 ml. The serum / virus mixtures were incubated for 2 hours at 37 ° C and 5% CO ² , to allow neutralization of the virus, and then 25 ml of this mixture (containing approximately 115 pfu) was inoculated into duplicate wells of cells HEp-2 in 96-well plates. After 2 h at 37 ° C and 5% CO2, the cells are superimposed on 0.75% methylcellulose containing medium with 5% and incubated for 40-46 hours. The number of syncytia was enumerated by immunostaining with monoclonal antibodies specific for fusion between RSV and nucleoproteins (AbD Serotec) followed by automatic counting (CTL Immunospot S5 UV analyzer). The neutralization titer is defined as the inverse of the serum dilution that produces at least a 60% reduction in the number of syncytiums per well, compared to controls (without serum). If the title of a sample is below the first serum dilution analyzed, ¹ : ^{2 0} , a ^{1: 1 0} titre was assigned for the purpose of calculating the group mean.

2. Results and conclusions

To model the use of the F subunit of the vaccine to increase serum neutralization titers in adult humans previously infected with RSV, groups of 7 cotton rats or adult mice were inoculated with RSV and im immunized with subunit vaccine. RSV F with or without adjuvant 7 weeks later (cotton rats) 11 and 15 weeks later (mice). The results of these experiments demonstrate that the subunit vaccine v Rs F effectively reinforces serum specific IgG F and the neutralizing RSV responses primed by RSV F infection in cotton mice and rats (Tables 1 and 2). The times of reinforcement in the response of antibodies specific to RSV F result in vaccination with the VRS F subunit was greater in mice (10 to 50 times increase in neutralization titres and 9 to 51 times increase in the specific IgG titer of F) than in cotton rats (4 to ⁶ times the increase in the neutralization titer and 7 to 25 times the increase in the specific IgG titer of F), but in both species the increase in the title it was very independent of the doses of antigen and adjuvant evaluated (Tables 1 and 2).

Table 1: Immunogenicity of the VRS F subunit vaccine in seropositive cotton rats for RSV

Fc-specific IgG title VRS neutralization title serum Gr. Inferí ³ Vaccinationb ⁷ sem 2 sem 4 sem 7 sem 2 sem after (sem 0) (sem 7) after the after the after after the __________________________________ infection _____ vaccination the infection infection vaccination

A VRS - 2.90 ± 0.19 2.80 ± 0.33 2.69 ± 0.03 2.44 ± 0.06 2.71 ± 0.03 B VRS ^{5 | jg of}

_{subunit F} 2.80 ± 0.35 4.16 ± 0.16 2.81 ± 0.08 2.64 ± 0.22 3.36 ± 0.13 5 jg of

C VRS subunit 2.93 ± 0.33 4.32 ± 0.18 2.64 ± 0.13 2.65 ± 0.05 3.39 ± 0.10

F / MF59

0.5 jg of

D VRS subunit 2.92 ± 0.24 3.99 ± 0.19 2.47 ± 0.02 2.67 ± 0.14 3.36 ± 0.05

F / MF59

0.05 jg of

E VRS subunit 2.90 ± 0.23 3.87 ± 0.17 2.68 ± 0.07 2.47 ± 0.09 3.25 ± 0.08

F / MF59

5 jg of

F VRS subunit F / 3.24 ± 0.52 4.07 ± 0.14 2.80 ± 0.20 2.64 ± 0.13 3.24 ± 0.11 alum

0.5 jg of

G VRS subunit 2.79 ± 0.12 3.92 ± 0.16 2.61 ± 0.09 2.39 ± 0.11 3.04 ± 0.06

F / alum

0.05 jg of

H VRS subunit 3.06 ± 0.17 4.05 ± 0.22 2.80 ± 0.17 2.62 ± 0.06 3.24 ± 0.22

F / alum

I - - n.p. n.p. 1.00 1.32 1.00 Abbreviations: Gr - group, sem - week, n.p. - not tested

at 1x105 pfu of VRS i.n.

b i.m.

c average of the log ^-10 of the title ± standard deviation of 7 individual animals per group

d average of log ¹⁰ of titles ± range of 2 combinations of 3-4 animals per group (1 combination of 2 animals in Gr. I) ___________________________________________________________________________

Example 3: Protection by passive transfer of antibodies derived from cotton rats and mice infected with RSV reinforced with F subunit vaccine

1. Procedures

Serum transfer and RSV infection of cotton rats and mice

Female cotton rats ( Sigmodon hispidis) were obtained from Harran Laboratories and female BALB / c mice were obtained from Charles River Laboratories. All studies were approved and performed according to the Novartis Animal Care and Use Committee. Groups of 7-8 adult female cotton rats were inoculated intraperitoneally (ip) (10 ml / kg) with one to three combinations of undiluted cotton rat serum: 1) "normal" - unexposed cotton rats To treatment collected, 2) "VSR" - cotton rats were infected with 1x105 pfu of RSV and serum was collected 7 weeks later, or 3) "VSR subunit" - cotton rats were infected with 1x105 pfu of RSV and vaccinated 7 weeks later with the F subunit. Serum was collected 2 to 4 weeks after vaccination with the F subunit (combined from animals vaccinated with 0.05 µg, 0.5 µg, or 5 µg subunit F, without adjuvant, or with alum adjuvant or MF59, analogously to the cotton rat experiment described in Example 2). An additional group of animals was injected with 10 ml / kg of a 1: 3 dilution of the serum combination "VSR subunit".

Groups of 4-8 BALB / c mice were inoculated i.p. (20 ml / kg) with one of two undiluted mouse serum combinations: 1) "normal" - collected from BALB / c mice not exposed to treatment, or 2) "RSV subunit" - BALB / c were infected with 1x106 pfu of RSV and were vaccinated 7 and 11 weeks later with the F subunit. The serum was collected 2 to 4 weeks after the 1st vaccination and 2 weeks after the 2 'vaccination and combined (the animals were vaccinated with 0, 05 jg, 0.5 jg, or 5 jg of subunit F, without adjuvant, or with alum adjuvant or MF59, analogously to the mouse experiment described in Example 2). An additional group of mice was injected with 20 ml / kg of a 1: 3 dilution of the serum combination "VSR subunit".

Two days after the transfer of serum to all cotton rats and mice, together with the untreated control animals, they were infected intranasally with RSV (1x105 pfu for cotton rats and 3x106 pfu for mice).

VSR plate test

The burden of RSV in the trans lung infection was determined by plaque assays. The lungs were collected and spent 4 days (mice) or 5 days (cotton rats) after RSV infection and a right lobe was inserted in the middle with 25% sucrose and disturbed with a tissue homogenizer. Cell-free supernatants from these samples were stored at -80 ° C. To analyze the presence and infectious viruses, dilutions of the clarified pulmonary homogenate (in PBS with 5% HI-FBS) were inoculated on monolayers of confluent Hep-2 cells in a volume of 200 ml / well of a 12-well plate. After 2 hours with gentle periodic stirring (37 ° C, 5% CO ² ), the inoculum was removed, and the cells were superimposed with 1.5 ml of 1.25% SeaPlaque agarose in medium supplemented with a 5% HI-FBS, glutamine, and antibiotics. After 3-4 days of incubation, the cells were superimposed with 1 ml of 1.25% SeaPlaque agarose in medium containing 0.1% black red (Sigma). The plates were counted a day later with the help of a light box. If the viral load of an individual animal was below the limit of detection of the assay (~ 200 pfu / g of lung), a titer of 100 pfu / g of lung was assigned for the purpose of calculating the group mean.

Neutralization test of VSV and F-specific IgG according to ELISA

These tests were carried out as described in Example 2.

2. Results and conclusions

To model the protection of human babies through passive transfer of antibodies with an increase in neutralizing titres from their mothers previously infected with RSV and with reinforcement of the F subunit, the inventors infected adult cotton rats with RSV, some of them received a booster with the VSR F subunit vaccine 7 weeks later, the serum was collected, and the serum was injected ip to cotton rats or adult mice not infected with non-immunized RSV (4-8 animals per group, cotton rat serum was injected into cotton rats, and mouse serum was injected into mice). Two days after serum transfer, all animals were stimulated with RSV i.n.

The neutralizing activity of RSV could easily be detected in the serum of recipient rodents one day after passive transfer of donor serum (Tables 3 and 4). Rodents receiving serum from RSV-infected donors immunized with the F subunit vaccine had the highest serum RSV neutralization titres after transfer and the lowest viral load in the lung after challenge with the RSV, when they are compared with rodents that received serum from donors infected with RSV but not immunized, normal serum, or did not receive serum before stimulation with RSV (Tables 3 and 4). These data illustrate that passively transferred serum can protect from stimulation with RSV, and that the higher the neutralization titre of the transferred serum, the greater the protection against RSV (Tables 3 and 4 and Figs. 1A and 1B). Since the receivers of serum transferred from cotton rats infected with RSV immunized with the F subunit vaccine, but not from cotton rats infected with RSV but not immunized with the F subunit vaccine, were protected from stimulation with RSV, these Results support the protection of human babies born to pregnant women (who have inevitably suffered from RSV infections in the past) immunized with the VRS F subunit vaccine.

To determine the half-life of the RSV-specific antibody after passive transfer, rodent groups received injections i.p. with undiluted serum from RSV-infected rodents immunized with the F subunit vaccine, and the RSV neutralization titer in the receptors was measured at several time points later. The results of these studies demonstrate that the half-life of passively transferred antibodies was approximately 2 weeks in both cotton rats and mice (Tables 5 and 6). Therefore, every two-fold increase in the neutralization titer above the threshold of protection by the VRS F subunit vaccine should extend the protection in passive antibody receptors by 2 weeks, in these rodent models.

Table 3: Protection by passive transfer of serum from cotton rats infected with RSV immunized with the F subunit vaccine

1 day after serum transfer ^{5 days after} Serum transferred to Stimb b _{stimulus with RSV Gr.}

(day 0) (day 2) _IgG title Neutralization title Viral load of the lung specific to HRV of serum _RSV

A VRS subunit VRS 2.81 ± 0.09 2.52 ± 0.09 3.18 ± 0.42

B ^{1: 3 VRS}

VRS _subunit 2.43 ± 0.30 2.16 ± 0.12 5.36 ± 0.47

C VRS VRS 1.64 ± 0.05 1.92 ± 0.15 5.48 ± 0.23

Normal D RSV 0.70 ± 0.00 1.04 ± 0.12 5.82 ± 0.24

E - RSV 0.70 ± 0.00 1.00 ± 0.00 5.74 ± 0.35

Abbreviations: Gr - group, stim.- stimulus

a combined serum of cotton rats infected with RSV immunized with the subunit vaccine ("RSV subunit"), not vaccinated ("RSV"), or not exposed to treatment ("normal"). 10 ml / kg of undiluted or diluted 1: 3 serum, as indicated, i.p. in groups of recipient cotton rats.

b 1x105 pfu of VRS i.n.

average c_d of the log ^-10 of the title ± standard deviation of 7-8 individual animals per group

e log-iQ average pfu / g lung ± standard deviation of 7-8 individual animals per group

Table 4: Protection by passive transfer of serum from BALB / c mice infected with RSV immunized with the F subunit vaccine

1 day after serum transfer ^{4 days after the} transferred Serum (Stim.b day _{stimulus with RSV Gr.}

0) ^{(day 2)} IgG title Neutralization title Viral load of RSV specific serum séricod pulmóne

A VRS subunit VRS 4.85 ± 0.04 2.90 ± 0.06 2.65 ± 0.07 B 1: 3 VRS subunit VRS 4.30 ± 0.05 2.20 ± 0.20 4.78 ± 0.39

Normal ^D RSV 0.70 ± 0.00 1.00 ± 0.00 4.95 ± 0.37

E - RSV 0.70 ± 0.00 1.04 ± 0.11 4.96 ± 0.26

Abbreviations: Gr - group, stim.- stimulus

a combined serum of BALB / c mice infected with RSV reinforced with the F subunit vaccine ("RSV subunit") or not exposed to treatment ("normal"). 20 ml / kg of undiluted or diluted 1: 3 serum, as indicated, i.p. to groups of recipient mice.

b 3x106 pfu of VRS i.n.

average c_d of the log ^-10 of the title ± standard deviation of 4-8 individual animals per group

e log average ^-10 pfu / g lung ± standard deviation of 4-8 individual animals per group

Table 5: Decrease in serum RSV neutralization titres in cotton rats receiving passive antibodies

Day after transfer a VRS neutralization title serum b Half-life

1 2.51 ± 0.00

7 2.27 ± 0.01

14 2.24 ± 0.05

19 2.11 ± 0.14

34 1.84 ± 0.08

15 days

48 1.47 ± 0.06

10 ml / kg of undiluted combined serum of cotton rats infected with RSV immunized with the F subunit vaccine ("RSV subunit") were injected i.p. to a group of 8 cotton rats

b average of the log-ip title ± range of 2 combinations of 4 animals__________________________________

Table 6: Decrease in serum RSV neutralization titers in passive antibody receptor mice

Day after transfer a VRS neutralization title serum b Half-life

1 2.87 ± 0.01

9 2.65 ± 0.04

16 2.43 ± 0.03

23 2.26 ± 0.08

36 2.08 ± 0.02

44 1.83 ± 0.04 ^{14 days}

a20 ml / kg of undiluted combined serum of BALB / c mice infected with RSV immunized with the F subunit vaccine ("RSV subunit") were injected i.p. to a group of 13 BALB / c mice.

b average of the log-io title ± SEM of 2-3 combinations of 4-5 animals

Example 4: Induction of a protective immune response by the RSV SAM ™ vaccine in rodents in the presence or absence of passively transferred RSV immune serum

1. Procedures

RSV SAM ™ vaccine

In these studies an RSV SAM ™ vaccine (Self-amplification message) (RNA replicon vaccine) encoding the full length VRS F protein with a deletion in the fusion peptide region was used. RNA was formulated with a cationic nanoemulsion (CNE, 4.4 mg / ml DOTAP, 0.5% SPAN 85, 0.5% Tween 80, 4.3% squalene in a 10 M citrate buffer pH 6, 5 in aqueous phase) before injection. The CNE was prepared analogously to the loaded MF59 described above (Ott et al., Journal of Controlled Release, volume 79, pages 1-5, 2002), with a major modification. DOTAP dissolved directly in squalene, and no organic solvent was used.

The RNA was diluted to a concentration of 300 pg / ml in a 2 M citrate buffer containing 20 M NaCl and 560 M sucrose. The RNA was added directly in an equivalent volume of CNE with gentle vortexing. The solution was allowed to stand at room temperature for 1 hour. After complexation, the resulting solution was diluted to the appropriate concentration and used within 1 hour.

Serum transfer, vaccination, and infection of rodents by RSV

Pure serum or a 1: 3 dilution of serum from female cotton rats that had been infected with RSV and had received a booster with the SAM VRS vaccine had ip (10 mg / kg) injected into groups of 16 cotton rats non-immunized adults not exposed to RSV. Groups of control cotton rats were injected ip with undiluted normal cotton rat serum or left untreated. Half of the animals in each group were vaccinated im with 15 pg of the RSV SAM vaccine (100 pl of total volume in 1 posterior limb) on days 2 and 20 of the study. All cotton rats were stimulated in with 1x105 pfu of RSV on day 49, and 5 days later, spleens and lungs were collected for T lymphocyte analysis and viral load. A similar study was conducted with BALB / c mice with passive transfer of mouse serum immunized with RSV or normal followed by vaccination of a subset of each group with 15 pg of the SAM VRS vaccine (100 pl of total volume divided between 2 hind limbs ), except that the serum dose was 20 ml / kg, the SAM vs vaccines were administered on days 2 and 22, the groups included 5 additional animals, whose spleens were collected on day 36 for the analysis of F-specific T lymphocytes mice they were stimulated with 3x106 pfu of RSV on day 45, and lungs and spleens were collected on day 49. The combinations of cotton rat and mouse serum used for transfer in these studies were identical to those used in Example 3.

Neutralization test of VSV and F-specific IgG according to ELISA

These tests were carried out as described in Example 2.

RSV test on plates

This test was performed as described in Example 3.

Functional T lymphocyte assay in cotton rat: cellular ELISA for secreted cytokines

IFNy and antigen-specific IL-4 secretion in cotton rats was measured by ELISA using R&D Systems kits with the following protocol modifications. Splenocytes (3-4 x 105 nucleated living cells) in 200 ml of serum-free T lymphocyte media (RPMI, 10 M HEPES, 100 jM non-essential amino acids, 50 jM 2-mercaptoethanol, and antibiotics) were added to cytokine capture antibody coated plates. The cells were stimulated with a cocktail of 5 µM each of the three VRS F peptides (F29-43, F49-63 and F247-261). The recombinant cytokine patterns were diluted in 1% FBS T lymphocyte media. After 24 hours, the secreted cytokines were detected according to the manufacturer's recommendations. The results are reported as pg pure / ml (average of triplicate wells with stimulus - average of wells without triplicate stimulation).

Functional T lymphocyte assay in mice: intracellular cytokine staining and flow cytometry

Spleens were collected on the indicated study days and processed to monocellular suspensions. When the splenocytes of the individual animals were analyzed separately, 1 culture stimulated with antigen and one culture not stimulated for each animal were established, and when the splenocytes of all the animals in a group were combined and analyzed in combination, 2 were established. cultures stimulated with antigen and two cultures not stimulated for each combination. Antigen-stimulated cultures contained 1x106 splenocytes, peptides 85-93 of VRS F and 249-258 (each at 1 M), peptides of VRS F 51-66, 164-178, and 309-323 ((each at 5 M), mAb against CD28 (1 jg / ml), and Brefeldin A (1: 1000) The non-stimulated cultures did not contain the VRS F peptides, and on the other hand were identical to the stimulated cultures. hours at 37 ° C, the cultures were processed for immunofluorescence.The cells were washed and then stained with fluorescently labeled monoclonal antibodies (mAb) directed against fluorescently labeled CD4 and CD8.The cells were washed again and then fixed with Cytofix / Cytoperm for 20 minutes, then the fixed cells were washed with Perm wash buffer and then stained with fluorescently labeled mAbs specific for IFNy, TNFa, IL-2 and IL-5. The stained cells were washed and then analyzed in a LSR II flow cytometer The F software lowJo was used to analyze the acquired data. The subsets of CD4 + 8- and CD8 + 4- T lymphocytes were analyzed separately. For each subset of a given sample, the percentage of cells positive for cytokines was determined. The percentage of VRS F antigen-specific T lymphocytes was calculated as the difference between the% of cytokine-positive cells in antigen stimulated cultures and the percentage of cytokine-positive cells in non-stimulated cultures.

2. Results and conclusions

To model the use of the SAM VRS vaccine in infants not exposed to RSV, the inventors evaluated the immunogenicity and efficacy of the SAM VRS vaccine in cotton rats and mice not exposed to RSV. The SAM VRS vaccine was immunogenic in both species, triggering VRS serum neutralization titres of 1: 776 in cotton rats and 1: 209 in mice, when measured 3-4 weeks after the 2nd i.m. vaccination. of 15 jg of the SAM VRS vaccine (Table 7 and 8, groups H). In mice, the VRS sAm vaccine triggered a strong F-specific CD8 + T lymphocyte response and a moderate Th4 CD4 + T lymphocyte response, characterized by the production of IFNy, TNFa, and IL-2 without significant IL-5 production ( Table 9, group H). In both species, the SAM RSV RNA vaccine provided almost complete protection against a nasal stimulus with RSV administered 3-4 weeks after the 2nd RNA vaccination, as can be seen by a greater reduction of 99.9% in pulmonary viral loads, compared to unimmunized control animals (Tables 7 and 8, group H compared to group D).

RSV neutralizing antibodies were passively transferred from the mother to the young through the placenta and protected the young from severe RSV disease during the first months of life; however, these same antibodies can also interfere with the active immune response against the SAM VRS vaccine. Therefore, the inventors also investigated whether the immunogenicity and efficacy of the SAM VRS vaccine in rodents was affected by passively acquired RSV-specific antibodies. RSV-neutralizing and F-specific IgG responses could be rapidly detected in the serum of recipient rodents one day after passive transfer of pure or diluted 1: 3 serum from RSV-infected rodents who received a vaccine booster the VRS F subunit (Tables 7-8). Forty-eight days after transferring the serum of immunity against RSV, the neutralization titers of RSV fell to very low or undetectable levels in non-vaccinated recipient cotton rats, pro were> 1:50 in those vaccinated with Rn (Table 7, groups A and B in comparison to groups E and F). These data demonstrate that the SAM VRS vaccine was able to induce a neutralizing RSV response in the presence of passively transferred immune serum. However, the neutralizing response induced by the SAM VRS vaccine was approximately 10 times less in the presence of passive RSV specific antibody than in the absence of this antibody (Table 7, groups E and F compared to groups G and H). In Balb / c mice, the RSV neutralizing titres measured after the second vaccination with SAM RSV were similar in the presence or absence of passively transferred RSV specific antibody (Table 8, groups E and F compared to groups G and H). Although these data suggest that the passively transferred antibody does not inhibit the antibody response induced by the SAM VRS vaccine in mice, it should be noted that the neutralization titer 44 days after serum transfer in mice vaccinated with SAM VRS was a combination of the title induced by the SAM VRS vaccine and the one derived from passive transfer (the neutralizing antibody was still detected in mice not vaccinated at this time point). Therefore, passively transferred RSV-specific antibody is likely to inhibit the antibody response induced by the SAM RSV vaccine to some extent in mice as well.

Although passively transferred RSV-specific antibody partially suppressed the antibody response induced by SAM RSV in rodents, it does not suppress the T-lymphocyte response induced by SAM RSV. In cotton rats, the magnitude of the splenic F-specific IFNy response 5 days after RSV infection was equivalent in animals vaccinated with SAM SAMs regardless of whether or not the animals had received a serum dose before vaccination (Table 10, group E compared to H). The results were similar in mice, where F-specific CD4 + and CD8 + T splenic responses measured 14 days after the second vaccination with SAM RSV, or 4 days after the second vaccination with SAM RSV were equivalent in the presence or absence of serum passively transferred (Tables 9 and 11, group E compared to G and H).

To determine whether the SAM VRS vaccine was capable of inducing a protective immune response in the presence of passive antibody from donors infected with RSV who received a booster with the F subunit vaccine, cotton rats or mice were inoculated with the serum of immunity against RSV, they were vaccinated twice with the SAM VRS vaccine, and then infected with RSV 3-4 weeks later. Four to five days after infection, the pulmonary viral load of these animals was at least 1.4 log-io lower than in control animals that did not receive serum or RNA before RSV infection (Tables 7 and 8, groups E and F compared to D). Since the passively transferred antibody had been metabolized at that time and, therefore, that the animals were no longer protecting the stimulus with v Rs (Tables 7 and 8, groups A and B), any protective effect can be attributed to the SAM VRS vaccine. These data demonstrate, therefore, that the SAM v Rs vaccine is capable of inducing a protective immune response in the presence of the passively transferred RSV-specific antibody. However, the protective efficacy of the SAM RSV vaccine was greater in the absence of specific serum from passive RSV (Tables 7 and 8, groups G and H).

In conclusion, these results support the use of the VRS SAM vaccine in children born to mothers vaccinated with the VRS F subunit vaccine, as they provide preclinical evidence in two rodent models that the SAM VRS vaccine is capable of inducing a response protective immune system in the presence of the RSV specific antibody passively transferred. Although the RSV neutralization response induced by the SAM VRS vaccine was partially suppressed by the passive antibody, the T lymphocyte response did not remain and, therefore, the possible protective mechanism was cell-mediated immunity.

As summarized in Table 12, the data in Examples 2-4 support the double vaccination strategy with RSV; only those cotton rats that received a serum dose of RSV-infected donors immunized with the F subunit vaccine on day 0, and subsequently vaccinated with the SAM VRS vaccine on days 2 and 20, were protected from an early stimulus (day 2) and late (day 49) with RSV.

Table 10: Responses of F-specific T lymphocytes after stimulation with RSV of cotton rats vaccinated with the SAM RSV vaccine in the presence or absence of passive-transferred RSV-specific antibody

Specific secreted cytokine of F 5 days later Gr. ^{Transfer of} Vacc.b (d 2 and 20) Estim.c (d of the stimulus with RSV (d 54) d _{serum (d 0)} 49)

IFNy IL-4 A VRS subunit - VRS 342 ± 138 2.6 ± 0.8

B ^{1: 3 VRS}

_subunit - VRS 1412 ± 404 3.0 ± 0.8 C normal - VRS 176 ± 45 1.0 ± 0.6

D - - VRS 860 ± 187 0.6 ± 0.6

E VRS subunit VRS SAM VRS 2941 ± 482 5.4 ± 0.2

F ^{1: 3 VRS}

SAM VRS _subunit VRS 2505 ± 606 3.4 ± 0.7 G normal VRS SAM VRS 4444 ± 1224 5.0 ± 2.8

H - VRS SAM VRS 3011 ± 461 2.8 ± 1.4

Abbreviations: Gr - group, d - day, vacc - vaccination, stimulus - stimulus

a combined serum of cotton rats infected with RSV reinforced with the subunit vaccine ("RSV subunit"), or not exposed to treatment ("normal"). 10 ml / kg of undiluted or diluted 1: 3 serum, as indicated, i.p. to groups of recipient cotton rats

b 15 | jg of VRS SAM i.m. vaccine or without vaccination

c 1x105 pfu of VRS i.n.

d average pg pure / ml ± standard error of the average of 4-5 individual animals (spleens) per group___________

Claims (14)

1. An inductive composition of an immune response against respiratory syncytial virus (RSV) that provides one or more RSV antigens for use in a procedure to provide protective immunity against RSV or the protection of a disease caused by RSV in a baby, in which the procedure comprises administering to the baby at 4 months of age or younger, the inductive composition of the immune response directed against RSV, in which said baby was a newborn of a woman to whom it was administered a composition that provides one or more RSV antigens that induced an immune response directed against RSV during the time said woman was pregnant with said baby, in which the one or more RSV antigens comprise a RSV F glycoprotein. 2. The composition for use according to claim 1, wherein the inductive composition of an immune response directed against RSV administered to said baby is sensitization or reinforcement. 3. The composition for use according to any one of claims 1 to 2, wherein the inducing composition of an immune response directed against RSV administered to said woman is administered during the second or third trimester of pregnancy. 4. The composition for use according to any one of claims 1 to 3, wherein administration to said baby occurs at one or more times selected from the group consisting of immediately after birth, 2 weeks after birth. Birth, 4 weeks after birth, 6 weeks after birth, 2 months after birth, 3 months after birth, and 4 months after birth. 5. The composition for use according to any one of claims 1 to 4, wherein the inductive composition of an immune response directed against RSV that was administered to the woman and the inductive composition of an immune response directed against the RSV administered to the baby are the same. 6. The composition for use according to any one of claims 1 to 4, wherein the inductive composition of an immune response directed against RSV that was administered to the woman and the inductive composition of an immune response directed against the RSV administered to the baby are not equal. 7. The composition for use according to any of the preceding claims, wherein the inductive composition of an immune response directed against RSV administered to the baby comprises a subunit composition of RSV, a nucleic acid, a recombinant viral vector, or a particle of a viral replicon (VRP). 8. The composition for use according to any one of claims 1 to 7, wherein the inducing composition of an immune response directed against RSV administered to said baby comprises a nucleic acid. 9. The composition for use according to claim 1 to 7, wherein the inducing composition of an immune response directed against RSV administered to women comprises a subunit composition of RSV. 10. The composition for use according to any one of claims 1 to 7, wherein the inducing composition of an immune response directed against RSV administered to the baby comprises a particle of a viral replicon. 11. The composition for use according to claim 7 or 9, wherein the composition of the RSV subunit comprises: (a) one or more mutations that prevent cleavage at one or both of the furin cleavage sites of a VRS F protein; and / or (b) one or more mutations in the fusion peptide region of a VRS protein. 12. The composition for use according to claim 7 or 8, wherein the nucleic acid comprises: (a) DNA; or (b) RNA. 13. The composition for use according to claim 12 wherein the RNA is a self-replicating RNA. 14. The composition for use according to claim 7 or 9, wherein the viral replicon particle comprises an alphavirus VRP.